Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

2012 ◽  
Vol 64 (2) ◽  
pp. 299-336 ◽  
Author(s):  
Martin J. Lohse ◽  
Susanne Nuber ◽  
Carsten Hoffmann
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Bioluminescence Resonance Energy Transfer ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based β-Arrestin2 Recruitment Assay

2005 ◽  
Vol 10 (5) ◽  
pp. 463-475 ◽  
Author(s):  
Fadi F. Hamdan ◽  
Martin Audet ◽  
Philippe Garneau ◽  
Jerry Pelletier ◽  
Michel Bouvier
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Bioluminescence Resonance Energy Transfer ◽  
G Protein Coupled

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of β-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with β-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- β-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and β-arrestin2- Renillaluciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. Atotal of 26,000 compounds were screened for inhibition of the agonist-promoted β-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced β-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1- ßarrestin recruitment assay in stablemammalian cells and its successful application in HTS for GPCRs antagonists.

scholarly journals Minireview: GPCR and G Proteins: Drug Efficacy and Activation in Live Cells

2009 ◽  
Vol 23 (5) ◽  
pp. 590-599 ◽  
Author(s):  
Jean-Pierre Vilardaga ◽  
Moritz Bünemann ◽  
Timothy N. Feinstein ◽  
Nevin Lambert ◽  
Viacheslav O. Nikolaev ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
G Proteins ◽  
Intracellular Signaling ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Live Cells ◽  
Mechanical Stimuli ◽  
G Protein Coupled

Abstract Many biochemical pathways are driven by G protein-coupled receptors, cell surface proteins that convert the binding of extracellular chemical, sensory, and mechanical stimuli into cellular signals. Their interaction with various ligands triggers receptor activation that typically couples to and activates heterotrimeric G proteins, which in turn control the propagation of secondary messenger molecules (e.g. cAMP) involved in critically important physiological processes (e.g. heart beat). Successful transfer of information from ligand binding events to intracellular signaling cascades involves a dynamic interplay between ligands, receptors, and G proteins. The development of Förster resonance energy transfer and bioluminescence resonance energy transfer-based methods has now permitted the kinetic analysis of initial steps involved in G protein-coupled receptor-mediated signaling in live cells and in systems as diverse as neurotransmitter and hormone signaling. The direct measurement of ligand efficacy at the level of the receptor by Förster resonance energy transfer is also now possible and allows intrinsic efficacies of clinical drugs to be linked with the effect of receptor polymorphisms.

scholarly journals Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling

2016 ◽  
Vol 473 (22) ◽  
pp. 4173-4192 ◽  
Author(s):  
Diana Zindel ◽  
Sandra Engel ◽  
Andrew R. Bottrill ◽  
Jean-Philippe Pin ◽  
Laurent Prézeau ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Mutational Analysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Parathyroid Hormone Receptor ◽  
Extracellular Signal ◽  
G Protein Coupled

The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489–Ser495 and Ser501–Thr506) specifically responsible for the majority of PTH(1–34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1–34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489–Ser495 and the second cluster Ser501–Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.

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