Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β ₂ AR

Abstract

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCRtargeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β 2 -adrenergic receptor (β 2 AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.signal transduction mechanisms | agonists and inverse agonists | conformational polymorphism | single-molecule fluorescence spectroscopy | phospholipid nanodiscs G protein-coupled receptors (GPCRs) mediate a multitude of physiological functions and are the targets for a myriad of drugs (1), many of which elicit different functional outcomes through the same receptor (2). It remains to be rationalized at the molecular level why some drugs stimulate the signaling activity of a GPCR (full or partial agonists), whereas others either repress the receptor (inverse agonists) or have no effect on the intrinsic signaling activity (neutral antagonists). Moreover, the existence of a high basal activity of some GPCRs (3) suggests that the conformational transitions leading to activation may occur spontaneously, even in the absence of ligands, which in turn raises questions about the mechanistic roles of GPCR ligands. Understanding the mechanisms and pathways of receptor activation or deactivation, and how these are linked to the binding of ligands with different chemical structures and pharmacological efficacies, will aid in design of new GPCR-targeted drugs with tailored pharmacological responses and fewer side effects. To attain these goals, new methods are required to visualize the conformational dynamics of GPCRs in the presence and absence of drugs.The β 2 -adrenergic receptor (β 2 AR) has been extensively investigated in crystals (4, 5), by NMR in solution (6-11), by bulk fluorescence spectroscopy in s...

Function-specific virtual screening for GPCR ligands using a combined scoring method

“…It should be noted that the β 2 R was also the ideal receptor for such an analysis, as much information is available for this receptor including (and most importantly) multiple crystal structures in different activation states and in complex with both agonists and antagonists/inverse agonists59262728293031323334. Generally, aminergic GPCRs have a deep and well-defined binding site without a large solvent-exposed area which make them suitable for docking simulations as demonstrated by other successful prospective structure-based virtual screening studies against aminergic GPCR crystal structures6174144454648.…”

β 2 -Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop

“…This apparent discrepancy can be explained by the different availability of cysteine groups for labeling with sulfhydryl-reactive reagents in the two experiments. From experience with 19 F-NMR studies of TET-labeled β 2 AR (Horst et al, 2013; Liu et al, 2012a) and with single-molecule fluorescence studies of Cy3-labeled β 2 AR (Lamichhane et al, 2015), it is now known that the three cysteines C265, C327, and C341 all react with the labeling reagents. Therefore, single-cysteine variants of β 2 AR were used here and in our earlier work (Liu et al, 2012a) to obtain spectra from individually labeled cysteines (Figures 1 and 2).…”

What Do Structures Tell Us About Chemokine Receptor Function and Antagonism?

“…from the extracellular ligand binding site to the intracellular G protein-coupling interface) and “downside up”. For example, addition of an extracellular agonist to β 2 AR results in conformational changes at the intracellular ends of receptor TM helices (45, 71, 107), and enhances the stability of receptor:G protein complexes (144) (“upside-down”). On the other hand, addition of either G protein or a G protein-mimicking nanobody Nb80 (124), both of which bind intracellularly, affects affinity and binding kinetics of extracellular agonists (30, 107) (“downside-up”).…”