Conformational Profiling of the 5-HT_2A Receptor Using FlAsH BRET

Abstract

Functional selectivity or biased agonism describes ligand-specific activation of particular downstream signaling pathways following drug treatment. This phenomenon may be exploited for the development of drugs with increased target selectivity and consequently better safety profiles. Analyzing bias at the level of signal transduction pathways is challenging due to cell type-dependent differences in the expression or activity of downstream effector proteins. Bioluminescence resonance energy transfer (BRET) has previously been used to characterize biased agonism at the level of the receptor using fluorescent biarsenical hairpin (FlAsH) binders as energy acceptors. By walking the FlAsH binding tetracysteine tag into different positions within the intracellular loops and carboxyl terminus of the 5-HT_2A receptor along with a C-terminally fused Renilla luciferase, we generated a panel of seven conformation-sensitive biosensors that were able to capture conformational information in response to agonist. These FlAsH BRET-based biosensors were expressed in HEK 293 cells but also in the more relevant N2A cells, a neuronal-like cell line where 5-HT_2A receptor biology can be captured. These biosensors were first validated for cell surface expression and normal signaling phenotypes, followed by conformational analysis in response to the full agonist 5-HT. These results demonstrate a potentially simple tool for drug discovery once conformational profiles are correlated with their downstream signaling pathways.