Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy

Olofsson, Linnéa; Felekyan, Suren; Doumazane, Etienne; Scholler, Pauline; Fabre, Ludovic; Zwier, Jurriaan M.; Rondard, Philippe; Seidel, Claus A. M.; Pin, Jean‐Philippe; Margeat, Emmanuel

doi:10.1038/ncomms6206

Cited by 95 publications

(127 citation statements)

References 59 publications

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“…Our ability to detect spontaneous transitions between two intrinsic receptor conformations is likely due to the unique photophysical properties of Cy3, the nativelike environment of the phospholipid nanodiscs, and the relatively long time scale of our observations. Recently, single-molecule fluorescence spectroscopy was used to visualize conformational transitions in the extracellular ligand-binding domain of the metabotropic glutamate receptor, either as an isolated protein dimer (28) or as part of the full-length receptor (29). In contrast, our observations of β 2 AR in nanodiscs reveal the dynamics of the membrane-embedded region of a GPCR.…”

Section: Discussioncontrasting

confidence: 47%

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

Lamichhane

Liu

Pljevaljčić

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

120

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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...

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Section: Discussioncontrasting

confidence: 47%

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

Lamichhane

Liu

Pljevaljčić

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

120

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“…In line with this concept, partial agonists would stabilize fewer receptors in active states, and concomitantly a greater fraction of partial agonist-bound receptors would remain in inactive states (22,24). However, it is puzzling how agonistbound receptors can adopt active and inactive states.…”

mentioning

confidence: 82%

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

Böck

Bermudez

Krebs

et al. 2016

Journal of Biological Chemistry

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G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood.Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M 2 receptors to demonstrate the existence and function of such inactive agonist⅐receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist⅐receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist⅐receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.Specific and coordinated cell-to-cell communication regulates the flow of information between cells, and proper information processing ensures physiological functions of biological systems. G protein-coupled receptors (GPCRs), 8 constituting the largest class of membrane proteins in mammals, are essential mediators of chemically and light-encoded information (1-4). GPCRs sense a great variety of extracellular stimuli, e.g. neurotransmitters and hormones, and subsequently translate this information into an intracellular response via G proteins, ␤-arrestins, and possibly GPCR-interacting proteins (2-5). Because of their abundance and relevance in regulating the majority of (patho-)physiological processes in humans, GPCRs have for a long time represented the most important drug targets being addressed by at least a third of all currently marketed drugs (6, 7).Agonist binding leads to receptor activation, which is followed by intracellular G protein recruitment and subsequent cell signaling. Breakthroughs in GPCR crystallography have led to inactive and active crystal structures of the same receptor protein. Among these are rhodopsin (8 -10) and more recently the ␤ 2 -adrenergic (11-14), M 2 muscarinic (15, 16), and -opioid receptors (17, 18). These structures most likely represent energetically favored, relatively stable inactive and active receptor⅐ligand complexes. Despite the diversity of crystallized receptors, a common...

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Section: Temporal Aspects-kinetics Along the Signaling Chainmentioning

confidence: 95%

“…Rapid dynamic transitions between different states are beginning to be observed for nonrhodopsin GPCRs, using a variety of methods including NMR and molecular dynamics simulations (Nygaard et al, 2013;Olofsson et al, 2014;Manglik et al, 2015). In contrast with this view, a recent study employing fluorescence correlation spectroscopy on soluble extracellular domains of a metabotropic glutamate receptor suggests that these receptors (or domains) may switch between only two conformations, termed open and closed (Olofsson et al, 2014). Overall, the transitions of GPCRs into truly active conformations appear to be incomplete under physiologic conditions, and stabilization by binding to a G-protein or to a (b-)arrestin appears to be required in order to produce a fully active receptor (Elgeti et al, 2013;Manglik et al, 2015).…”

Section: Temporal Aspects-kinetics Along the Signaling Chainmentioning

confidence: 99%

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

Lohse

Hofmann

2015

Mol Pharmacol

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Signaling by G-protein-coupled receptors is often considered a uniform process, whereby a homogeneously activated proportion of randomly distributed receptors are activated under equilibrium conditions and produce homogeneous, steady-state intracellular signals. While this may be the case in some biologic systems, the example of rhodopsin with its strictly local singlequantum mode of function shows that homogeneity in space and time cannot be a general property of G-protein-coupled systems. Recent work has now revealed many other systems where such simplicity does not prevail. Instead, a plethora of mechanisms allows much more complex patterns of receptor activation and signaling: different mechanisms of proteinprotein interaction; temporal changes under nonequilibrium conditions; localized receptor activation; and localized second messenger generation and degradation-all of which shape receptor-generated signals and permit the creation of multiple signal types. Here, we review the evidence for such pleiotropic receptor signaling in space and time.

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Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy

Cited by 95 publications

References 59 publications

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

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

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

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Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy

Cited by 95 publications

References 59 publications

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

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

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

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

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