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Review
.2022 Dec;12(12):e1124.
doi: 10.1002/ctm2.1124.

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

Affiliations
Review

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

Kayla E Kroning et al. Clin Transl Med.2022 Dec.

Abstract

G-protein-coupled receptors (GPCRs) are the most abundant receptor type in the human body and are responsible for regulating many physiological processes, such as sensation, cognition, muscle contraction and metabolism. Further, GPCRs are widely expressed in the brain where their agonists make up a large number of neurotransmitters and neuromodulators. Due to the importance of GPCRs in human physiology, genetically encoded sensors have been engineered to detect GPCR agonists at cellular resolution in vivo. These sensors can be placed into two main categories: those that offer real-time information on the signalling dynamics of GPCR agonists and those that integrate the GPCR agonist signal into a permanent, quantifiable mark that can be used to detect GPCR agonist localisation in a large brain area. In this review, we discuss the various designs of real-time and integration sensors, their advantages and limitations, and some in vivo applications. We also discuss the potential of using real-time and integrator sensors together to identify neuronal circuits affected by endogenous GPCR agonists and perform detailed characterisations of the spatiotemporal dynamics of GPCR agonist release in those circuits. By using these sensors together, the overall knowledge of GPCR-mediated signalling can be expanded.

Keywords: GPCR; genetically encoded sensor; neuromodulation; neurotransmission.

© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.

PubMed Disclaimer

Conflict of interest statement

The authors declare they have no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Schematic of G‐protein‐coupled receptor (GPCR) signalling cascade. Agonist binding causes a conformational change in the GPCR, which recruits G‐proteins and/or β‐arrestin to the GPCR. G‐protein binding leads to a change in concentration of second messengers and β‐arrestin binding causes internalisation of the GPCR. These agonist‐induced changes can be utilised in sensors, where they can cause the change of fluorescence, an enzymatic readout, or the transcription of a reporter gene.
FIGURE 2
FIGURE 2
Integrators versus real‐time sensors. Integrators leave a permeant mark in the cells exposed to G‐protein‐coupled receptor (GPCR) agonists, so large brain regions can be analysed at cellular resolution. Real‐time sensors can be used to observe the real‐time agonist‐induced neuronal activity.
FIGURE 3
FIGURE 3
Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET)‐based sensors. (A) Resonance energy transfer (RET)‐based sensor, where one RET pair is attached to the Gα‐protein and the other is attached to the G‐protein‐coupled receptor (GPCR). GPCR activationrecruits the G‐proteins, increasing the energy transfer efficiency. Acc, RET acceptor. (B) RET‐based sensor, where one RET pair is attached to the Gα‐protein mimic, such as a miniG protein or a nanobody, and the other is attached to the GPCR. GPCR activation recruits the G‐protein mimic, increasing the energy transfer efficiency. (C) Schematic of BERKY. BRET pairs are separated by an ER/K α‐helix linker, where the acceptor is at the end of the linker and the donor is at the start of the linker, fused to the membrane. KB‐1753, a peptide that binds to the active Gα‐protein, is attached to the acceptor pair. Agonist results in G‐protein activation which results in KB‐1753 binding and an increase in energy transfer efficiency. (D) RET‐based sensor, where one RET pair is attached to the Gα‐protein and the other is attached to the Gβ‐ and γ‐proteins. GPCR activation causes the distance to increase between the Gα and Gβγ proteins, decreasing the energy transfer efficiency. (E) RET‐based sensor, where one RET pair is attached to β‐arrestin and the other is attached to the GPCR. GPCR activation recruits β‐arrestin, increasing the energy transfer efficiency. (F) RET‐based sensor, where RET pairs are attached to either terminus of β‐arrestin. Agonist activation of the GPCR causes a conformational change in β‐arrestin, where there is an increase in energy transfer efficiency. (G) RET‐based sensor, where one RET pair is attached to the third intracellular loop of the GPCR and the other is attached to the GPCR's C‐terminal tail. Agonist‐induced activation causes a conformational change in the GPCR, thereby changing the energy transfer efficiency.
FIGURE 4
FIGURE 4
Periplasmic binding protein (PBP)‐based tools. (A) Resonance energy transfer pairs are attached to PBPs. Ligand binding induces a conformational change in the PBP, which results in a change in energy transfer efficiency. (B) PBPs are attached to either terminus of circularly permuted green fluorescent protein (cpGFP), where ligand binding to the PBP causes a conformational change in cpGFP, resulting in a fluorescence change.
FIGURE 5
FIGURE 5
Circularly permuted green fluorescent protein (cpGFP) inserted into the third intracellular loop of a G‐protein‐coupled receptor (GPCR). Upon agonist‐induced GPCR activation, the third intracellular loop of the GPCR undergoes a conformational change, changing the fluorophore environment of cpGFP and resulting in a fluorescence change.
FIGURE 6
FIGURE 6
Sensors that detect secondary messengers. (A) Calcium‐sensing proteins, calmodulin and M13, are attached to either terminus of circularly permuted green fluorescent protein (cpGFP). In the presence of calcium, calmodulin and M13 interact, resulting in a change in the fluorophore environment of cpGFP and a fluorescence increase. (B) Schematic of CAMYEL. Bioluminescence resonance energy transfer (BRET) pairs RLuc and yellow fluorescent protein (YFP) are tethered to EPAC. cAMP binding causes a conformational change in EPAC, where the energy transfer efficiency is decreased. (C) EPAC was attached to either terminus of cpGFP. cAMP‐induced EPAC conformational change results in a change in cpGFP fluorescence. (D) PKCγ, which only binds to diacylglycerol (DAG), is tethered to either terminus of cpGFP. DAG induces a conformational change in PKCγ, changing the cpGFP fluorescence.
FIGURE 7
FIGURE 7
Integrators. (A) Schematic of Tango. The C‐terminus of the G‐protein‐coupled receptor (GPCR) is fused with a tobacco etch virus protease cleavage site (TEVcs) and transcription factor (TF). β‐Arrestin is tethered to a TEV protease. Agonist binding to the GPCR recruits β‐arrestin, where the TEV protease can then cut at the TEVcs, releasing the TF so it can translocate to the nucleus and activate a reporter gene. (B) Schematic ofiTango2. Same basic mechanism as Tango, except the TEV protease is split into two components (TEV‐N and TEV‐C) that are fused to β‐arrestin and the GPCR. Additionally, the light oxygen voltage sensing (AsLOV2) domain cages the TEVcs. Agonist recruits β‐arrestin fused split TEV to the GPCR, where the split protease components can reassociate and light uncages TEVcs, allowing the protease to cut and release the TF. (C) Schematic of SPARK. Same basic mechanism asiTango2, except the TEV protease is not split, a truncated protease is used instead. Additionally, evolved LOV (eLOV) is used instead of AsLOV2. (D) Schematic of SPOTIT. Circularly permuted green fluorescent protein (cpGFP) and Nb39 are tethered to the C‐terminus of the GPCR. Nb39 inhibits cpGFP fluorophore formation. Agonist activates the opioid receptor (OR), recruiting Nb39 to the third intracellular loop, releasing cpGFP and allowing the fluorophore to form. (E) Schematic of SPOTon. Same basic mechanism of SPOTIT, except cpGFP‐Nb39 is fused to FK506 binding protein (FKBP) and the OR is fused to FKBP‐rapamycin binding domain (FRB). Rapamycin induces heterodimerisation of FKBP and FRB, bringing cpGFP‐Nb39 to the OR. Opioid activates the OR, recruiting Nb39 to the third intracellular loop and allowing the cpGFP fluorophore to mature. (F) Schematic of Trio. GFP is split into three component: β1‐9, β10 and β11. β10 is attached to β‐arrestin, β11 is attached to the GPCR and β1‐9 is expressed in the cytosol. Agonist‐induced GPCR activation recruits β‐arrestin to the C‐terminus of the GPCR, allowing the three split components of GFP to re‐associate and a fluorescence increase.
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