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.

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 Multiple GPCR Functional Assays Based on Resonance Energy Transfer Sensors

2021 ◽  
Vol 9 ◽  
Author(s):  
Yiwei Zhou ◽  
Jiyong Meng ◽  
Chanjuan Xu ◽  
Jianfeng Liu
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Functional Assays ◽  
High Throughput Drug Screening ◽  
Gpcr Activation ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) represent one of the largest membrane protein families that participate in various physiological and pathological activities. Accumulating structural evidences have revealed how GPCR activation induces conformational changes to accommodate the downstream G protein or β-arrestin. Multiple GPCR functional assays have been developed based on Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) sensors to monitor the conformational changes in GPCRs, GPCR/G proteins, or GPCR/β-arrestin, especially over the past two decades. Here, we will summarize how these sensors have been optimized to increase the sensitivity and compatibility for application in different GPCR classes using various labeling strategies, meanwhile provide multiple solutions in functional assays for high-throughput drug screening.

scholarly journals Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

2017 ◽  
Author(s):  
Marieke Mastop ◽  
Daphne S. Bindels ◽  
Nathan C. Shaner ◽  
Marten Postma ◽  
Theodorus W. J. Gadella ◽  
...  
Keyword(s):  
G Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Tandem Fusion ◽  
Red Fluorescent Proteins

AbstractGenetically encoded Förster Resonance Energy Transfer (FRET) based biosensors report on changes in biochemical states in single living cells. The performance of biosensors depends on their brightness and dynamic range, which are dependent on the characteristics of the fluorescent proteins that are employed. Cyan fluorescent protein (CFP) is frequently combined with yellow fluorescent protein (YFP) as FRET pair in biosensors. However, current YFPs are prone to photobleaching and pH changes. In addition, more efficient acceptors may yield biosensors that have higher contrast. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mChery and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. The sensor with mScarlet-I as acceptor and mTurquoise2 as donor shows a higher dynamic range in ratiometric FRET imaging experiments and less variability than with mCherry as acceptor, due to the high quantum yield and efficient maturation of mScarlet-I. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.

Quantum Dots and FRET-Nanobeads for Probing Genes, Proteins, and Drug Targets in Single Cells

2003 ◽  
Author(s):  
Amit Agrawal ◽  
Xiaohu Gao ◽  
Nitin Nitin ◽  
Gang Bao ◽  
Shuming Nie
Keyword(s):  
Quantum Dots ◽  
Drug Targets ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Length Scale ◽  
Imaging And Spectroscopy

Quantum dots are tiny light-emitting particles on the length scale of 2–10 nm, and FRET-nanobeads for fluorophore-embedded nanoparticles on the length scale of 40–200 nm based on the phenomenon of fluorescence resonance energy transfer (FRET). These materials are emerging as a new class of biological labels with properties and applications that are not available with traditional organic dyes and fluorescent proteins. In this ASME contribution, we report new developments in using semiconductor quantum dots for quantitative imaging and spectroscopy of single cancer cells. We also show results from intracellular staining of actin filaments using FRET-nanobeads. These results raise new possibilities in disease diagnostics, drug and biochemical discovery, cancer imaging, molecular profiling, and disease staging.

FZD5 is a Gαq-coupled receptor that exhibits the functional hallmarks of prototypical GPCRs

2018 ◽  
Vol 11 (559) ◽  
pp. eaar5536 ◽  
Author(s):  
Shane C. Wright ◽  
Maria Consuelo Alonso Cañizal ◽  
Tobias Benkel ◽  
Katharina Simon ◽  
Christian Le Gouill ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Group Of Seven ◽  
Stem Cell Regulation ◽  
Wnt Proteins ◽  
Gpcr Activation ◽  
Class B ◽  
Activation Profile ◽  
Functional Inactivation ◽  
Class F

Frizzleds (FZDs) are a group of seven transmembrane–spanning (7TM) receptors that belong to class F of the G protein–coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD5) is one of the most studied class F GPCRs that promote the functional inactivation of the β-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)–based assays that demonstrated that FZD5 activated Gαq and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.

scholarly journals Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3105 ◽  
Author(s):  
Henning Höfig ◽  
Michele Cerminara ◽  
Ilona Ritter ◽  
Antonie Schöne ◽  
Martina Pohl ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strong Increase ◽  
Single Molecule Level ◽  
Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

scholarly journals Proteasomal conformation controls unfolding ability

2021 ◽  
Vol 118 (25) ◽  
pp. e2101004118
Author(s):  
Julianna R. Cresti ◽  
Abramo J. Manfredonia ◽  
Christopher E. Bragança ◽  
Joseph A. Boscia ◽  
Christina M. Hurley ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational State ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Ubiquitinated Protein ◽  
Equilibrium Conditions ◽  
Substrate Processing

The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome’s various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome’s ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome’s conformational state and its unfolding ability.

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