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

Seungwoo Chang, Elliott M. Ross

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article numbere45651
JournalPloS one
Volume7
Issue number9
DOIs
StatePublished - Sep 20 2012

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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