scholarly journals GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells

2020 ◽  
Vol 117 (25) ◽  
pp. 14522-14531
Author(s):  
Allison Anderson ◽  
Ikuo Masuho ◽  
Ezequiel Marron Fernandez de Velasco ◽  
Atsushi Nakano ◽  
Lutz Birnbaumer ◽  
...  
Keyword(s):  
G Protein ◽  
Coupling Efficiency ◽  
Resonance Energy Transfer ◽  
Cardiac Pacemaker ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Protein Isoforms ◽  
Minimal Impact ◽  
Signaling Dynamics ◽  
The Impact

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+(GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαoover Gαias a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαoor Gαi2was consistent with these findings. Gαi2ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαoablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαosubstrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

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.

scholarly journals Agonist-induced formation of unproductive receptor-G12complexes

2020 ◽  
Vol 117 (35) ◽  
pp. 21723-21730
Author(s):  
Najeah Okashah ◽  
Shane C. Wright ◽  
Kouki Kawakami ◽  
Signe Mathiasen ◽  
Joris Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Receptor Internalization ◽  
Protein Association ◽  
Protein Activation ◽  
Protein Receptor ◽  
G Protein Activation

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2receptors (V2R) associate with both Gsand G12heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gscomplexes, V2R-G12complexes are not destabilized by guanine nucleotides and do not promote G12activation. Activating V2R does not lead to signaling responses downstream of G12activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12heterotrimers. Overexpressing G12inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12that are insensitive to nucleotides, suggesting that unproductive GPCR-G12complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

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.

Homogeneous GTP Binding Assay Employing QRET Technology

2010 ◽  
Vol 15 (3) ◽  
pp. 261-267 ◽  
Author(s):  
Anita Rozwandowicz-Jansen ◽  
Jonne Laurila ◽  
Eija Martikkala ◽  
Heini Frang ◽  
Ilkka Hemmilä ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Binding Assay ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Fluorescent Detection ◽  
Time Resolved ◽  
Gtp Binding ◽  
Gtpγs Binding

Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5′-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [35S]GTPγS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a non–fluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonhydrolyzable GTP analog (Eu-GTP). The quenching resonance energy transfer (QRET) method relies on the use of Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the radiolabel [35S]GTPγS assay. Upon activation of recombinant human α2A-adrenoceptors (α2A-AR) expressed in Chinese hamster ovary cells, guanosine-5′-diphosphate is released from the α-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of α2A-AR with 5 different α2-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [35S]GTPγS and heterogeneous Eu-GTP filtration assays. Equal potencies and efficacy rank orders were observed in all 3 assays but with a lower signal-to-background ratio and increased assay variation in the QRET assay compared to the Eu-GTP filtration and the nonhomogeneous [35S]GTPγS binding assays.

Kinetics of G-protein-coupled receptor signalling and desensitization

2004 ◽  
Vol 32 (6) ◽  
pp. 1029-1031 ◽  
Author(s):  
C. Krasel ◽  
J.-P. Vilardaga ◽  
M. Bünemann ◽  
M.J. Lohse
Keyword(s):  
G Protein ◽  
Second Messengers ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
G Protein Coupled Receptor ◽  
Protein Subunit ◽  
Protein Activation ◽  
G Protein Coupled ◽  
Kinetics Of

The kinetics of G-protein-coupled receptor activation and deactivation has, so far, been measured only indirectly, most frequently by assessing the production of various second messengers. We have developed methods based on fluorescence resonance energy transfer to quantify the kinetics of receptor activation by agonist (measured as conformational change in the receptor), the kinetics of G-protein activation (measured as G-protein subunit rearrangement) and the kinetics of receptor inactivation by arrestins (measured as receptor–arrestin interaction). Using these methods, we show that receptor activation by agonists and signalling to G-proteins occur on the subsecond time scale, whereas receptor desensitization is limited by receptor phosphorylation and proceeds more slowly.

scholarly journals A FRET based biosensor for measuring Gα13 activation in single cells

2017 ◽  
Author(s):  
Marieke Mastop ◽  
Nathalie R. Reinhard ◽  
Cristiane R. Zuconelli ◽  
Fenna Terwey ◽  
Theodorus W. J. Gadella ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Coupling Efficiency ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protease Activated Receptors ◽  
Heterotrimeric G Protein ◽  
Gpcr Activation ◽  
P115 Rhogef

AbstractFörster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human Gα13 subunit. Three variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged Gα13 subunits were used as FRET donor and combined with cp173Venus fused to the Gγ2 subunit as the acceptor. We constructed Gα13 biosensors by generating a single plasmid that produces Gα13-mTurquoise2, Gβ1 and cp173Venus-Gγ2. The Gα13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were treated with thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the Gα13 sensor could be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the Gα13 biosensor is a valuable tool for live-cell measurements that probe Gα13 activation.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

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