Genetically encoded tools for in vivo G‐protein‐coupled receptor agonist detection at cellular resolution

Kroning, Kayla E.; Wang, Wenjing

doi:10.1002/ctm2.1124

Cited by 7 publications

(4 citation statements)

References 137 publications

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“…Similarly, the split-galactosidase PathHunter assay requires a unique cell line for each β-arrestin isoform and cannot report dynamics. Alternative β-arrestin recruitment architectures also include enzyme-induced cleavage of transcription factors and bioluminescent resonance energy transfer (BRET)-based assays (Kroning and Wang, 2022). For example, the cleavage-dependent Tango assay system was recently extended to include β-arrestin 1 (Zeghal et al, 2023).…”

Section: Discussionmentioning

confidence: 99%

ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment

French,

Meqbil,

van Rijn

2023

Front. Pharmacol.

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Background: Modern methods for quantifying signaling bias at G protein–coupled receptors (GPCRs) rely on using a single β-arrestin isoform. However, it is increasingly appreciated that the two β-arrestin isoforms have unique roles, requiring the ability to assess β-arrestin isoform preference. Thus, methods are needed to efficiently screen the recruitment of both β-arrestin isoforms as they compete for a target GPCR in cells.Methods: We used molecular cloning to develop fusion proteins of the δ-opioid receptor (δOR), β-arrestin 1, and β-arrestin 2 to fragments of click beetle green and click beetle red luciferases. In this assay architecture, recruitment of either β-arrestin 1 or 2 to the δOR generates a spectrally distinct bioluminescent signal, allowing us to co-transfect all three constructs into cells prior to agonist challenge.Results: We demonstrate that our new assay, named “ClickArr,” is a live-cell assay that simultaneously reports the recruitment of both β-arrestin isoforms as they compete for interaction with the δOR. We further find that the partial δOR agonist TAN67 has a significant efficacy bias for β-arrestin 2 over β-arrestin 1 when recruitment is normalized to the reference agonist leu-enkephalin. We confirm that ClickArr reports this bias when run either as a high-throughput endpoint or high-throughput kinetic assay, and cross-validate this result using the PathHunter assay, an orthogonal commercial assay for reporting β-arrestin recruitment to the δOR.Conclusion: Our results suggest that agonist:GPCR complexes can have relative β-arrestin isoform bias, a novel signaling bias that may potentially open up a new dimension for drug development.

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Section: Discussionmentioning

confidence: 99%

ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment

French,

Meqbil,

van Rijn

2023

Front. Pharmacol.

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“…Therefore, there is a need for further development of integrators for detecting neuromodulators. My lab has so far designed two new classes of molecular integrators, which have the potential to be applicable for large brain volume detection in live animals, followed by post-mortem analysis. ,,,,− …”

Section: Sensors For Neuromodulatorsmentioning

confidence: 99%

Protein-Based Tools for Studying Neuromodulation

Wang

2024

ACS Chem. Biol.

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Neuromodulators play crucial roles in regulating neuronal activity and affecting various aspects of brain functions, including learning, memory, cognitive functions, emotional states, and pain modulation. In this Account, we describe our group's efforts in designing sensors and tools for studying neuromodulation. Our lab focuses on developing new classes of integrators that can detect neuromodulators across the whole brain while leaving a mark for further imaging analysis at high spatial resolution. Our lab also designed chemical-and light-dependent protein switches for controlling peptide activity to potentially modulate the endogenous receptors of the neuromodulatory system in order to study the causal effects of selective neuronal pathways.

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“…RET can be used for the detection of molecular proximity and conformational changes within a molecule at high temporal resolution. FRET and BRET have been reviewed multiple times elsewhere (Kauk & Hoffmann, 2018;Kroning & Wang, 2022;Olsen & English, 2023). Here we describe advanced RET applications, single-molecule, time-resolved, and fluorescence lifetime imaging FRET, which allow the observation of protein conformational changes and signalling dynamics in living samples with molecular resolution and structural insights (Figure 2 and 3, Supporting Table 1).…”

Section: Resonance Energy Transfer (Ret) Techniquesmentioning

confidence: 99%

Microscopy and spectroscopy approaches to study GPCR structure and function

Fessl

Majellaro²,

Bondar

2023

Preprint

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The GPCR signalling cascade is a key pathway responsible for the signal transduction of a multitude of physical and chemical stimuli, including light, odorants, neurotransmitters, and hormones. Understanding the structural and functional properties of the GPCR cascade requires direct observation of signalling processes in high spatial and temporal resolution with minimal perturbation to endogenous systems. Optical microscopy and spectroscopy techniques are uniquely suited to this purpose because they excel at multiple spatial and temporal scales and can be used in living objects. Here, we review recent developments in microscopy and spectroscopy technologies which enable new insights into GPCR signalling. We focus on advanced techniques with high spatial and temporal resolution, single-molecule methods, labelling strategies, and approaches suitable for endogenous systems and large living objects. This review aims to assist researchers in choosing appropriate microscopy and spectroscopy approaches for a variety of applications in the study of cellular signalling.

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Genetically encoded tools for in vivo G‐protein‐coupled receptor agonist detection at cellular resolution

Abstract: G-protein-coupled receptor (GPCR) agonist sensors utilise downstream GPCR signalling events, such as GPCR conformational changes and protein binding, to give a measurable sensor readout.

Cited by 7 publications

References 137 publications

ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment

ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment

Protein-Based Tools for Studying Neuromodulation

Microscopy and spectroscopy approaches to study GPCR structure and function

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