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

作者：Chang Seungwoo; Ross Elliott M^*

来源：PLos One, 2012, 7(9): e45651.

DOI：10.1371/journal.pone.0045651

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 Forster 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 G alpha(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 G alpha(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.

• 出版日期2012-9-20