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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 …