Resonance Energy Transfer-Based Approaches to Study GPCRs

Ayoub, Mohammed Akli

doi:10.1016/bs.mcb.2015.10.008

Cited by 14 publications

(18 citation statements)

References 228 publications

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“…However, eliminating the need for an external light source for donor excitation gives BRET some advantages over FRET: it does not cause photodamage to cells, photobleaching of fluorophores, background autofluorescence, or direct excitation of the acceptor (41). Thanks to these advantages, the BRET technique has been widely implemented for drug screening, especially in the G protein-coupled receptor research field (2). Implementation of high-throughput screening on ion channels, including TRPs, has proved more problematic (42).…”

Section: Discussionmentioning

confidence: 99%

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Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels

Ruigrok

Shahid

Goudeau

et al. 2017

Biophysical Journal

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Multiplexed bioluminescence resonance energy transfer (BRET) assays were developed to monitor the activation of several functional transient receptor potential (TRP) channels in live cells and in real time. We probed both TRPV1 intramolecular rearrangements and its interaction with Calmodulin (CaM) under activation by chemical agonists and temperature. Our BRET study also confirmed that: (1) capsaicin and heat promoted distinct transitions, independently coupled to channel gating, and that (2) TRPV1 and Ca-bound CaM but not Ca-free CaM were preassociated in resting live cells, while capsaicin activation induced both the formation of more TRPV1/CaM complexes and conformational changes. The BRET assay, based on the interaction with Calmodulin, was successfully extended to TRPV3 and TRPV4 channels. We therefore developed a full-spectral three-color BRET assay for analyzing the specific activation of each of the three TRPV channels in a single sample. Such key improvement in BRET measurement paves the way for the simultaneous monitoring of independent biological pathways in live cells.

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Section: Discussionmentioning

confidence: 99%

“…This is a system of choice for monitoring both constitutive and regulated inter-and intramolecular interaction. Among these techniques, bioluminescence resonance energy transfer (BRET) has become a popular, broadly applicable method, particularly useful in molecular pharmacology, especially concerning G proteincoupled receptors (2).…”

Section: Introductionmentioning

confidence: 99%

Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels

Ruigrok

Shahid

Goudeau

et al. 2017

Biophysical Journal

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“…Although approaches such as immunoblotting, cross-linking and co-immunoprecipitation have been employed to study the basis of GPCR dimerization/oligomerization, they have limitations for the study of interactions involving integral membrane proteins due to the use of non-physiological buffers and detergents that may cause either non-native aggregation or disruption of native biological interactions. Those limitations have been addressed with the development of biophysical methods based on resonance energy transfer (RET) between two molecules, known as the “donor” and “acceptor,” positioned within a restricted distance (in the region of 2–8 nm) and defined orientation ( Alvarez-Curto et al., 2010b , Ayoub and Pfleger, 2010 , Ayoub, 2016 ). These include both bioluminescence resonance energy transfer (BRET) and variants of fluorescence resonance energy transfer (FRET), and both have been widely applied to the study of protein-protein interactions and the dimerization of muscarinic receptors and other GPCRs in particular ( Goin and Nathanson, 2006 , McMillin et al., 2011 , Alvarez-Curto et al., 2010a , Ciruela et al., 2010 , Marsango et al., 2015a , Sposini et al., 2015 ).…”

Section: Muscarinic Acetylcholine Receptorsmentioning

confidence: 99%

Muscarinic receptor oligomerization

et al. 2018

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G protein-coupled receptors (GPCRs) have been classically described as monomeric entities that function by binding in a 1:1 stoichiometric ratio to both ligand and downstream signalling proteins. However, in recent years, a growing number of studies has supported the hypothesis that these receptors can interact to form dimers and higher order oligomers although the molecular basis for these interactions, the overall quaternary arrangements and the functional importance of GPCR oligomerization remain topics of intense speculation.Muscarinic acetylcholine receptors belong to class A of the GPCR family. Each muscarinic receptor subtype has its own particular distribution throughout the central and peripheral nervous systems. In the central nervous system, muscarinic receptors regulate several sensory, cognitive, and motor functions while, in the peripheral nervous system, they are involved in the regulation of heart rate, stimulation of glandular secretion and smooth muscle contraction. Muscarinic acetylcholine receptors have long been used as a model for the study of GPCR structure and function and to address aspects of GPCR dimerization using a broad range of approaches. In this review, the prevailing knowledge regarding the quaternary arrangement for the various muscarinic acetylcholine receptors has been summarized by discussing work ranging from initial results obtained using more traditional biochemical approaches to those generated with more modern biophysical techniques.This article is part of the Special Issue entitled ‘Neuropharmacology on Muscarinic Receptors’.

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“…More importantly, the therapeutic strategies involving RAS are mostly based on AngII receptor blockers combined with ACE inhibitors (Heyndrickx, 1993;Serruys et al, 1995;Pratt Richard and Dzau Victor, 1996;Tsukuda et al, 2011;Urushihara et al, 2011). Therefore, the objective of the present study was to investigate the putative functional interaction between AT1R and thrombin and its receptor, PAR1, in vitro by assessing the pharmacological and functional effects of thrombin and PAR1 on AT1R activation and signaling, internalization, and endosomal trafficking, as well as their physical interaction, in HEK293 cells using various BRET proximity-based assays as previously described (Ayoub, 2016).…”

Section: Introductionmentioning

confidence: 99%

Interplay Between Angiotensin II Type 1 Receptor and Thrombin Receptor Revealed by Bioluminescence Resonance Energy Transfer Assay

et al. 2020

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The key hormone of the renin-angiotensin system (RAS), angiotensin II (AngII), and thrombin are known to play major roles in the vascular system and its related disorders. Previous studies reported connections between AngII and thrombin in both physiological and pathophysiological models. However, the molecular mechanisms controlling such interplay at the level of their receptors belonging to the family of G protein-coupled receptors (GPCRs) are not fully understood. In this study, we investigated the functional interaction between the AngII type 1 receptor (AT1R) and the thrombin receptor [or protease-activated receptor 1 (PAR1)] in human embryonic kidney 293 (HEK293) cells. For this, we used various bioluminescence resonance energy transfer (BRET) proximitybased assays to profile the coupling to the heterotrimeric Gaq protein, b-arrestin recruitment, and receptor internalization and trafficking in intact cells. The overall doseresponse and real-time kinetic BRET data demonstrated the specific molecular proximity between AT1R and PAR1 resulting in their functional interaction. This was characterized by thrombin inducing BRET increase within AT1R/Gaq and AT1R/b-arrestin pairs and synergistic effects observed upon the concomitant activation of both receptors suggesting a positive allosteric interaction. The BRET data corroborated with the data on the downstream Gaq/inositol phosphate pathway. Moreover, the selective pharmacological blockade of the receptors revealed the implication of both AT1R and PAR1 protomers in such a synergistic interaction and the possible transactivation of AT1R by PAR1. Interestingly, the positive action of PAR1 on AT1R activation was contrasted with its apparent inhibition of AT1R internalization and its endosomal trafficking. Finally, BRET saturation and co-immunoprecipitation assays supported the physical AT1-PAR1 interaction in HEK293 cells. Our study reveals for the first time the functional interaction between AT1R and PAR1 in vitro characterized by a transactivation and positive allosteric modulation of AT1R and inhibition of its desensitization and internalization. This finding may constitute the molecular basis of the well-known interplay between RAS and thrombin. Thus, our data should lead to revising some findings on the implication of

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Resonance Energy Transfer-Based Approaches to Study GPCRs

Cited by 14 publications

References 228 publications

Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels

Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels

Muscarinic receptor oligomerization

Interplay Between Angiotensin II Type 1 Receptor and Thrombin Receptor Revealed by Bioluminescence Resonance Energy Transfer Assay

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