Distinct Conformational Dynamics of Three G Protein-Coupled Receptors Measured Using FlAsH-BRET Biosensors

A number of studies have profiled G protein-coupled receptor (GPCR) conformation using fluorescent biaresenical hairpin binders (FlAsH) as acceptors for BRET or FRET. These conformation-sensitive biosensors allow reporting of movements occurring on the intracellular surface of a receptor to investigate mechanisms of receptor activation and function. Here, we generated eight FlAsH-BRET-based biosensors within the sequence of the β₂-adrenergic receptor (β₂AR) and compared agonist-induced responses to the angiotensin II receptor type I (AT1R) and the prostaglandin F2α receptor (FP). Although all three receptors had FlAsH-binding sequences engineered into the third intracellular loops and carboxyl-terminal domain, both the magnitude and kinetics of the BRET responses to ligand were receptor-specific. Biosensors in ICL3 of both the AT1R and FP responded robustly when stimulated with their respective full agonists as opposed to the β₂AR where responses in the third intracellular loop were weak and transient when engaged by isoproterenol. C-tail sensors responses were more robust in the β₂AR and AT1R but not in FP. Even though GPCRs share the heptahelical topology and are expressed in the same cellular background, different receptors have unique conformational fingerprints.