Quantifying biased signaling in GPCRs using BRET-based biosensors

Namkung, Yoon; Radresa, Olivier; Armando, Sylvain; Devost, Dominic; Beautrait, Alexandre; Gouill, Christian Le; Laporte, Stéphane A.

doi:10.1016/j.ymeth.2015.04.010

Cited by 34 publications

(46 citation statements)

References 20 publications

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“…The generation of pcDNA3.1-myc-OTR-WT was as previously described (68). The polycistronic G␣ q activation sensor was used as previously described (69).…”

Section: Constructsmentioning

confidence: 99%

“…As mentioned above, we used an established polycistronic G␣ q activation sensor that is based on physical separation of G␣ and G␥ subunits following receptor activation (69). Briefly, cells in white 96-well plates (Corning), transfected with the biosensor, and GPCR of interest were washed once and then left with 80 l of Kreb's buffer in a white 96-well plate.…”

Section: G␣ Q -Activation Assaymentioning

confidence: 99%

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Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors

Sleno

Devost

Pétrin

et al. 2017

Journal of Biological Chemistry

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G protein-coupled receptors (GPCRs) are conformationally dynamic proteins transmitting ligand-encoded signals in multiple ways. This transmission is highly complex and achieved through induction of distinct GPCR conformations, which preferentially drive specific receptor-mediated signaling events. This conformational capacity can be further enlarged via allosteric effects between dimers, warranting further study of these effects. Using GPCR conformation-sensitive biosensors, we investigated allosterically induced conformational changes in the recently reported F prostanoid (FP)/angiotensin II type 1 receptor (AT1R) heterodimer. Ligand occupancy of the AT1R induced distinct conformational changes in FP compared with those driven by PGF2α in bioluminescence resonance energy transfer (BRET)-based FP biosensors engineered with luciferase (RLuc) as an energy donor in the C-tail and fluorescein arsenical hairpin binder (FlAsH)-labeled acceptors at different positions in the intracellular loops. We also found that this allosteric communication is mediated through Gα and may also involve proximal (phospholipase C) but not distal (protein kinase C) signaling partners. Interestingly, β-arrestin-biased AT1R agonists could also transmit a Gα-dependent signal to FP without activation of downstream Gα signaling. This transmission of information was specific to the AT1R/FP complex, as activation of Gα by the oxytocin receptor did not recapitulate the same phenomenon. Finally, information flow was asymmetric in the sense that FP activation had negligible effects on AT1R-based conformational biosensors. The identification of partner-induced GPCR conformations may help identify novel allosteric effects when investigating multiprotein receptor signaling complexes.

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“…The generation of pcDNA3.1-myc-OTR-WT was as previously described (68). The polycistronic G␣ q activation sensor was used as previously described (69).…”

Section: Constructsmentioning

confidence: 99%

Section: G␣ Q -Activation Assaymentioning

confidence: 99%

Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors

Sleno

Devost

Pétrin

et al. 2017

Journal of Biological Chemistry

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“…Upon binding agonists, GPCRs couple to single or multiple Gprotein subtypes and initiate cell-specific signaling pathways. Studies with novel bioluminescence resonance energy transfer sensors show GPCRs exhibit a promiscuous and "pluridimensional" behavior, coupling to many Gα-proteins with different strengths (1)(2)(3)(4). While a receptor may show similar affinity to different Gα proteins, the cellular context may render certain couplings moot (1,(5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

“…SPASM is sensitive to measuring weak and dynamic protein−protein interactions in cellular conditions (29,37,38). This permits comparison between the binding affinities of cognate (canonical signaling partners) and noncognate (weak or uncharacterized partners) Gα proteins at the same stoichiometric ratios with the GPCR, which is not feasible with other biophysical techniques used in live cells (1,4,39,40). Recent findings with SPASM FRET sensors show a physiologic effect of noncognate G proteins to prime GPCR signaling within the cell (41), demonstrating the importance for probing these noncognate G-protein interactions within the cell.…”

mentioning

confidence: 99%

Conformational plasticity of the intracellular cavity of GPCR−G-protein complexes leads to G-protein promiscuity and selectivity

Sandhu

Touma

Dysthe

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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While the dynamics of the intracellular surface in agonist-stimulated GPCRs is well studied, the impact of GPCR dynamics on G-protein selectivity remains unclear. Here, we combine molecular dynamics simulations with live-cell FRET and secondary messenger measurements, for 21 GPCR−G-protein combinations, to advance a dynamic model of the GPCR−G-protein interface. Our data show C terminus peptides of Gα s , Gα i , and Gα q proteins assume a small ensemble of unique orientations when coupled to their cognate GPCRs, similar to the variations observed in 3D structures of GPCR−G-protein complexes. The noncognate G proteins interface with latent intracellular GPCR cavities but dissociate due to weak and unstable interactions. Three predicted mutations in β 2 -adrenergic receptor stabilize binding of noncognate Gα q protein in its latent cavity, allowing promiscuous signaling through both Gα s and Gα q in a dose-dependent manner. This demonstrates that latent GPCR cavities can be evolved, by design or nature, to tune G-protein selectivity, giving insights to pluridimensional GPCR signaling.G-protein−coupled receptor | GPCR | functional selectivity | structural plasticity | dynamics Author contributions: M.S.

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“…BRET has been successfully applied to study GPCR dimerization (Angers et al ., 2000), and more recently to study proximal interactions of GPCRs with different signaling effectors including G proteins and β-arrestins (Shukla et al ., 2008; Molinari et al ., 2010; Ceraudo et al ., 2014). Recent new advances in BRET technology include the discovery of biosensors, which do not involve the labeling of receptor as either a BRET donor or acceptor, and allow the identification of biased signaling from unknown compounds for any GPCR of interest (Namkung et al ., 2016). …”

Section: Discovering Biased Signaling On Gpcrmentioning

confidence: 99%

Biased G Protein-Coupled Receptor Signaling: New Player in Modulating Physiology and Pathology

Bologna

Teoh

Bayoumi

et al. 2017

Biomolecules & Therapeutics

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G protein-coupled receptors (GPCRs) are a family of cell-surface proteins that play critical roles in regulating a variety of pathophysiological processes and thus are targeted by almost a third of currently available therapeutics. It was originally thought that GPCRs convert extracellular stimuli into intracellular signals through activating G proteins, whereas β-arrestins have important roles in internalization and desensitization of the receptor. Over the past decade, several novel functional aspects of β-arrestins in regulating GPCR signaling have been discovered. These previously unanticipated roles of β-arrestins to act as signal transducers and mediators of G protein-independent signaling have led to the concept of biased agonism. Biased GPCR ligands are able to engage with their target receptors in a manner that preferentially activates only G protein- or β-arrestin-mediated downstream signaling. This offers the potential for next generation drugs with high selectivity to therapeutically relevant GPCR signaling pathways. In this review, we provide a summary of the recent studies highlighting G protein- or β-arrestin-biased GPCR signaling and the effects of biased ligands on disease pathogenesis and regulation.

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Quantifying biased signaling in GPCRs using BRET-based biosensors

Cited by 34 publications

References 20 publications

Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors

Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors

Conformational plasticity of the intracellular cavity of GPCR−G-protein complexes leads to G-protein promiscuity and selectivity

Biased G Protein-Coupled Receptor Signaling: New Player in Modulating Physiology and Pathology

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