Studying Ligand Efficacy at G Protein-Coupled Receptors Using FRET
Drug “ligands” that bind G protein-coupled receptors (GPCRs) can either stimulate, fully (full agonists) or partially (partial agonists), or reduce (inverse agonists) basal receptor activity, by stabilizing different receptor conformations. The term “intrinsic efficacy” was introduced as a parameter to express the ability of a ligand to activate its receptor and to differentiate the varying signaling capacity of diverse ligands when they occupy the same fraction of a single receptor. Most methods use downstream biochemical and physiological responses as proxies of “intrinsic efficacy” but cannot measure it directly at the level of the receptor. Here I describe the development of a Förster resonance energy transfer (FRET) approach that permits the rigorous measurement of the intrinsic efficacy of a ligand directly at the level of a GPCR and independent from variation in experimental conditions. This approach also allows intrinsic efficacies of ligands to be linked with the effects of receptor polymorphisms or receptor heterodimerization.
Key wordsG protein-coupled receptor α2A-Adrenergic receptor β1-Adrenergic receptor GPCR heterodimer GPCR polymorphisms Heterotrimeric G proteins Förster resonance energy transfer Ligand efficacy Conformational changes FlAsH labeling
This work was supported by start-up funds from the Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine and by National Institutes of Health (NIH) grant DK087688. I thank Tim Feinstein for careful comments on this manuscript.
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