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

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.