Activation Biosensor for G Protein-Coupled Receptors: A FRET-Based m1 Muscarinic Activation Sensor That Regulates Gq

We describe the design, construction and validation of a fluorescence sensor to measure activation by agonist of the m1 muscarinic cholinergic receptor, a prototypical class I Gq-coupled receptor. The sensor uses an established general design in which Förster resonance energy transfer (FRET) from a...

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Bibliographic Details
Published in:PloS one 2012-09, Vol.7 (9), p.e45651
Main Authors: Chang, Seungwoo, Ross, Elliott M.
Format: Article
Language:English
Subjects:
Acetylcholine receptors
Acetylcholine receptors (muscarinic)
Activation
Biology
Biophysics
Biosensors
Chemistry
Deoxyribonucleic acid
DNA
Energy transfer
Fluorescein
Fluorescence
Fluorescence resonance energy transfer
G protein-coupled receptors
Medicine
Membranes
Proteins
Receptors
Sensors
Spectrum analysis
Studies
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Summary:We describe the design, construction and validation of a fluorescence sensor to measure activation by agonist of the m1 muscarinic cholinergic receptor, a prototypical class I Gq-coupled receptor. The sensor uses an established general design in which Förster resonance energy transfer (FRET) from a circularly permuted CFP mutant to FlAsH, a selectively reactive fluorescein, is decreased 15–20% upon binding of a full agonist. Notably, the sensor displays essentially wild-type capacity to catalyze activation of Gαq, and the purified and reconstituted sensor displays appropriate regulation of affinity for agonists by Gq. We describe the strategies used to increase the agonist-driven change in FRET while simultaneously maintaining regulatory interactions with Gαq, in the context of the known structures of Class I G protein-coupled receptors. The approach should be generally applicable to other Class I receptors which include numerous important drug targets.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0045651