Activation biosensor for G protein-coupled receptors: a FRET-based m1 muscarinic activation sensor that regulates...

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 G(q)-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 Gα(q), and the purified and reconstituted sensor displays appropriate regulation of affinity for agonists by G(q). We describe the strategies used to increase the agonist-driven change in FRET while simultaneously maintaining regulatory interactions with Gα(q), 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.