A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells
G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the c...
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| Format: | Online |
| Language: | English |
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Public Library of Science
2016
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4723041/ |
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pubmed-4723041 |
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pubmed-47230412016-01-30 A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells van Unen, Jakobus Stumpf, Anette D. Schmid, Benedikt Reinhard, Nathalie R. Hordijk, Peter L. Hoffmann, Carsten Gadella, Theodorus W. J. Goedhart, Joachim Research Article G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation. Public Library of Science 2016-01-22 /pmc/articles/PMC4723041/ /pubmed/26799488 http://dx.doi.org/10.1371/journal.pone.0146789 Text en © 2016 van Unen et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
van Unen, Jakobus Stumpf, Anette D. Schmid, Benedikt Reinhard, Nathalie R. Hordijk, Peter L. Hoffmann, Carsten Gadella, Theodorus W. J. Goedhart, Joachim |
| spellingShingle |
van Unen, Jakobus Stumpf, Anette D. Schmid, Benedikt Reinhard, Nathalie R. Hordijk, Peter L. Hoffmann, Carsten Gadella, Theodorus W. J. Goedhart, Joachim A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells |
| author_facet |
van Unen, Jakobus Stumpf, Anette D. Schmid, Benedikt Reinhard, Nathalie R. Hordijk, Peter L. Hoffmann, Carsten Gadella, Theodorus W. J. Goedhart, Joachim |
| author_sort |
van Unen, Jakobus |
| title |
A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells |
| title_short |
A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells |
| title_full |
A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells |
| title_fullStr |
A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells |
| title_full_unstemmed |
A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells |
| title_sort |
new generation of fret sensors for robust measurement of gαi1, gαi2 and gαi3 activation kinetics in single cells |
| description |
G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation. |
| publisher |
Public Library of Science |
| publishDate |
2016 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4723041/ |
| _version_ |
1613527333721866240 |