G protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-specific signaling responses remain poorly understood. The discovery of functional selectivity, or biased agonism, has fundamentally impacted our understanding of G protein-coupled receptor (GPCR) signaling. Biased agonists preferentially activate particular G protein-dependent or non-canonical G protein-independent pathways, including those mediated by arrestin recruitment to the receptor. In addition to their roles in GPCR desensitization and trafficking, arrestins also scaffold signaling pathways distinct from those involving G protein. Arrestin-biased ligands have been identified for a number of different receptors, including dopamine D2, angiotensin, and beta-2 (?2) adrenergic receptors. Carvedilol, for instance, is an arrestin-biased agonist for the ?1 and ?2 adrenergic receptors (?2AR) that exhibits beneficial cardioprotective effects. Arrestin-specific signaling is likely triggered by agonist-induced conformational changes within the receptor and the formation of agonist-dependent conformations of the GPCR-arrestin complex. Recent findings further suggest that arrestin may retain ?active-like? conformations even after dissociating from the receptor. Although arrestin-specific agonism plays a critical role in GPCR- mediated signaling, we lack a molecular and kinetic understanding of how GPCR-arrestin complexes form and the physical basis of biased agonism. Such information is paramount to the rational design of drugs with desired efficacies at specific effectors. Ensemble spectroscopic methods can potentially provide critical insights into GPCR-mediated signaling, but bulk methods of this kind rely on the interpretation of average responses from very large numbers of potentially heterogeneous receptor-effector complexes, masking critical information about the conformational changes underpinning function. We have therefore sought to establish the means to directly quantify the dynamics of individual GPCRs proteins at the single-molecule scale using wide-field, total internal reflection fluorescence (TIRF) single-molecule fluorescence and fluorescence resonance energy transfer (FRET) imaging methods. Single-molecule imaging offers the potential to bypass the limitations of ensemble measurements by enabling direct observations of stochastic, asynchronous conformational processes associated with function. Using this approach, we have quantified ligand efficacy for G protein coupling to the beta2-adrenergic receptor (?2AR). In the proposed initiatives we will delineate the agonist- dependent conformational changes associated with receptor and arrestin complex formation using the ?2AR as our model system. These efforts will reveal, for the first time, the order and timing of the key conformational transitions in both receptor and arrestin required for, and associated with, receptor-arrestin interaction, information vital to advancing our understanding of the arrestin activation mechanism and the nature of biased agonism with important ramifications for the development of more selective therapeutics.

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In the proposed research we will utilize single-molecule fluorescence and fluorescence energy transfer imaging methods to probe the mechanism of GPCR-mediated arrestin activation by small-molecule ligands. The goal of these investigations is to provide quantitative insights into the allosteric mechanism by which specific GPCR agonists can preferentially trigger arrestin activation and signaling pathways instead of activation of heterotrimeric G proteins. This feature of GPCR signaling, known as biased agonism or functional selectivity, is a critical determinant of ligand efficacy and the pharmacological consequences of broadly utilized clinical therapies.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
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Biophysics of Neural Systems Study Section (BPNS)
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Silberberg, Shai D
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Weill Medical College of Cornell University
Schools of Medicine
New York
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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