Application of fluorescence correlation spectroscopy to the measurement of agonist binding to a G-protein coupled receptor at the single cell level

2004 ◽  
Vol 126 ◽  
pp. 197 ◽  
Author(s):  
Stephen J. Briddon ◽  
Richard J. Middleton ◽  
Andrew S. Yates ◽  
Michael W. George ◽  
Barrie Kellam ◽  
...  
Keyword(s):  
Single Cell ◽  
G Protein ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Single Cell Level ◽  
Agonist Binding ◽  
G Protein Coupled Receptor ◽  
Cell Level ◽  
Fluorescence Correlation ◽  
G Protein Coupled

scholarly journals Single molecule binding of a ligand to a G-protein-coupled receptor in real time using fluorescence correlation spectroscopy, rendered possible by nano-encapsulation in styrene maleic acid lipid particles

Nanoscale ◽  
2020 ◽  
Vol 12 (21) ◽  
pp. 11518-11525 ◽  
Author(s):  
Rachael L. Grime ◽  
Joelle Goulding ◽  
Romez Uddin ◽  
Leigh A. Stoddart ◽  
Stephen J. Hill ◽  
...  
Keyword(s):  
Real Time ◽  
G Protein ◽  
Single Molecule ◽  
Maleic Acid ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptor ◽  
Fluorescence Correlation ◽  
Lipid Particles ◽  
G Protein Coupled

Combining the technologies of encapsulation of GPCRs in SMA lipid particles with fluorescence correlation spectroscopy provides a versatile characterisation platform.

scholarly journals Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging

2020 ◽  
Vol 21 (21) ◽  
pp. 7880
Author(s):  
Leonore Mensching ◽  
Sebastian Rading ◽  
Viacheslav Nikolaev ◽  
Meliha Karsak
Keyword(s):  
Single Cell ◽  
G Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Single Cell Level ◽  
Cb2 Receptor ◽  
Cell Level ◽  
Cb2 Receptors ◽  
G Protein Coupled

G-protein coupled cannabinoid CB2 receptor signaling and function is primarily mediated by its inhibitory effect on adenylate cyclase. The visualization and monitoring of agonist dependent dynamic 3′,5′-cyclic adenosine monophosphate (cAMP) signaling at the single cell level is still missing for CB2 receptors. This paper presents an application of a live cell imaging while using a Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, for quantification of cAMP. We established HEK293 cells stably co-expressing human CB2 and Epac1-camps and quantified cAMP responses upon Forskolin pre-stimulation, followed by treatment with the CB2 ligands JWH-133, HU308, β-caryophyllene, or 2-arachidonoylglycerol. We could identify cells showing either an agonist dependent CB2-response as expected, cells displaying no response, and cells with constitutive receptor activity. In Epac1-CB2-HEK293 responder cells, the terpenoid β-caryophyllene significantly modified the cAMP response through CB2. For all of the tested ligands, a relatively high proportion of cells with constitutively active CB2 receptors was identified. Our method enabled the visualization of intracellular dynamic cAMP responses to the stimuli at single cell level, providing insights into the nature of heterologous CB2 expression systems that contributes to the understanding of Gαi-mediated G-Protein coupled receptor (GPCR) signaling in living cells and opens up possibilities for future investigations of endogenous CB2 responses.

scholarly journals Effect of different agents onto multidrug resistant tumor cells revealed by fluorescence correlation spectroscopy

2008 ◽  
Vol 22 (4) ◽  
pp. 261-265
Author(s):  
Céline Boutin ◽  
Yann Roche ◽  
Christine Millot ◽  
Régis Deturche ◽  
JérÔme Plain ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Multidrug Resistant ◽  
Correlation Spectroscopy ◽  
The Other ◽  
Single Cell Level ◽  
Cell Level ◽  
Fluorescence Correlation ◽  
Plasma Membrane Fluidity ◽  
Cellular Medium

Fluorescence correlation spectroscopy (FCS) has been used to analyze the plasma membrane fluidity and heterogeneity of multidrug resistant cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been explored. Firstly, we reveal a modification of plasma membrane heterogeneities according to the addition of a fluidity modulator, benzyl alcohol. On the other hand, revertants such as verapamil and cyclosporin-A appear to act more specifically on the slow diffusion sites, such as lipids microdomains.

scholarly journals The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

2016 ◽  
Vol 44 (2) ◽  
pp. 624-629 ◽  
Author(s):  
Laura E. Kilpatrick ◽  
Stephen J. Hill
Keyword(s):  
G Protein ◽  
Fluorescence Correlation Spectroscopy ◽  
Photon Counting ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptors ◽  
Fluorescence Correlation ◽  
Membrane Bound ◽  
Molecular Brightness ◽  
G Protein Coupled

The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

The use of fluorescence correlation spectroscopy to characterise the molecular mobility of G protein-coupled receptors in membrane microdomains: an update

2021 ◽  
Vol 49 (4) ◽  
pp. 1547-1554
Author(s):  
Laura E. Kilpatrick ◽  
Stephen J. Hill
Keyword(s):  
G Protein ◽  
Fluorescence Correlation Spectroscopy ◽  
Molecular Mechanisms ◽  
Short Review ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptors ◽  
Membrane Microdomains ◽  
Sensitivity Threshold ◽  
Fluorescence Correlation ◽  
G Protein Coupled

It has become increasingly apparent that some G protein-coupled receptors (GPCRs) are not homogeneously expressed within the plasma membrane but may instead be organised within distinct signalling microdomains. These microdomains localise GPCRs in close proximity with other membrane proteins and intracellular signalling partners and could have profound implications for the spatial and temporal control of downstream signalling. In order to probe the molecular mechanisms that govern GPCR pharmacology within these domains, fluorescence techniques with effective single receptor sensitivity are required. Of these, fluorescence correlation spectroscopy (FCS) is a technique that meets this sensitivity threshold. This short review will provide an update of the recent uses of FCS based techniques in conjunction with GPCR subtype selective fluorescent ligands to characterise dynamic ligand–receptor interactions in whole cells and using purified GPCRs.

scholarly journals Sustained Activation of a G Protein-coupled Receptor via “Anchored” Agonist Binding

1996 ◽  
Vol 271 (39) ◽  
pp. 24029-24035 ◽  
Author(s):  
Stuart A. Green ◽  
Andrew P. Spasoff ◽  
Robert A. Coleman ◽  
Malcolm Johnson ◽  
Stephen B. Liggett
Keyword(s):  
G Protein ◽  
Agonist Binding ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Sustained Activation

scholarly journals Multiplexed fluorescence imaging of GPCR downstream signaling dynamics at the single-cell level

2021 ◽  
Author(s):  
Ryosuke Tany ◽  
Yuhei Goto ◽  
Yohei Kondo ◽  
Kazuhiro Aoki
Keyword(s):  
Single Cell ◽  
G Protein ◽  
Fluorescence Imaging ◽  
Imaging System ◽  
Single Cell Level ◽  
Cell Level ◽  
Gpcr Signaling ◽  
Downstream Signaling ◽  
Signaling Dynamics ◽  
Multiplexed Fluorescence Imaging

AbstractG-protein-coupled receptors (GPCRs) play an important role in sensing various extracellular stimuli, such as neurotransmitters, hormones, and tastants, and transducing the input information into the cell. While the human genome encodes more than 800 GPCR genes, only four Gα-proteins (Gαs, Gαi/o, Gαq/11, and Gα12/13) are known to couple with GPCRs. It remains unclear how such divergent GPCR information is translated into the downstream G-protein signaling dynamics. To answer this question, we report a multiplexed fluorescence imaging system for monitoring GPCR downstream signaling dynamics at the single-cell level. Genetically encoded biosensors for cAMP, Ca2+, RhoA, and ERK were selected as markers for GPCR downstream signaling, and were stably expressed in HeLa cells. GPCR was further transiently overexpressed in the cells. As a proof-of-concept, we visualized GPCR signaling dynamics of 5 dopamine receptors and 12 serotonin receptors, and found heterogeneity between GPCRs and between cells. Even when the same Gα proteins were known to be coupled, the patterns of dynamics in GPCR downstream signaling, including the signal strength and duration, were substantially distinct among GPCRs. These results suggest the importance of dynamical encoding in GPCR signaling.

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