An important advance in pharmacological understanding is that of functional selectivity, whereby different downstream effectors of a single G protein-coupled receptor (GPCR) can be differentially activated by distinct ligands. Interesting examples of functional selectivity have been identified in a number of different GPCRs, including dopamine receptors and opiate receptors, two key targets of drugs of abuse. Despite enormous recent advances in the structural biology of GPCRs, the detailed structural and kinetic mechanisms by which drug binding modulates receptor activity, and particularly why different drugs can lead to different effects at the same receptor, remain poorly understood. We propose a series of single-molecule fluorescence resonance energy transfer (smFRET) experiments on the beta2 adrenergic receptor (B2AR) to delineate the mechanistic basis for its differential activation by different ligands. SmFRET makes it possible to follow the movements of an individual molecule over time, providing a means of making direct measurements of the rates and amplitudes of transient changes in protein conformation during function. Data of this kind hold the promise of providing critical information for elucidating functional mechanisms, including the detection of static and dynamic heterogeneities and transiently populated, non-accumulating intermediates, mechanisms that are masked by ensemble methods. We propose the following specific aims: 1) To determine the impact of a range of ligands on the equilibrium distribution of the conformational states of the B2AR and on the transition rates between these states, as determined by smFRET. 2) To probe structural dynamics within the heterotrimeric G?s?? complex bound to agonist-activated B2AR using smFRET and to compare a spectrum of agonists to provide insights into their signaling differences. The platform of technologies we develop will help us to understand the distinctive molecular properties of different B2AR agonists and to formulate hypotheses for designing drugs with optimized properties. Additionally, these methods will be broadly applicable to studies of other GPCRs that are critical therapeutic targets in numerous diseases.

Public Health Relevance

We will use single-molecule imaging methods to study conformational changes within individual beta2 adrenergic receptors, a prototypical G protein-coupled receptor, in response to different drugs. This approach makes it possible to follow the dynamic movements of an individual receptor molecule over time, providing a means of understanding mechanistic differences between drugs and ultimately of improved drug design, with a long term goal of better understanding drugs of abuse and treatment of addiction.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Exploratory/Developmental Grants (R21)
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Biophysics of Neural Systems Study Section (BPNS)
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Rapaka, Rao
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Columbia University (N.Y.)
Schools of Medicine
New York
United States
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Gregorio, G Glenn; Masureel, Matthieu; Hilger, Daniel et al. (2017) Single-molecule analysis of ligand efficacy in ?2AR-G-protein activation. Nature 547:68-73