Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region

Kofuku, Yutaka; Ueda, Takumi; Okude, Junya; Shiraishi, Yutaro; Kondo, Kazunori; Maeda, Masahiro; Tsujishita, Hideki; Shimada, Ichio

doi:10.1038/ncomms2046

Cited by 184 publications

(217 citation statements)

References 40 publications

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“…Overall, in a liganded sample, we could resolve 20 peaks in the Ile d1 region of the spectrum, out of 29 Ile residues present in our construct (28 from the receptor and one from the C-terminal Protein C tag). The chemical shifts of the agonist-bound and inverse agonist-bound A 2A R are similar ( Figure 2A,B), showing subtle changes that are comparable in magnitude to those observed previously for 13 C-methyl-methionine probes in b 2 AR (Kofuku et al, 2014;Kofuku et al, 2012). The spectrum of the unliganded (apo) A 2A R shows a loss of most of the well-dispersed peaks present in the ligand-bound spectra ( Figure 2C), which could result from protein instability or conformational exchange on the ms-ms timescale.…”

Section: Resultssupporting

confidence: 58%

“…GPCR NMR studies have primarily used supplementation with labeled amino acids (Nygaard et al, 2013;Kofuku et al, 2014;Okude et al, 2015;Isogai et al, 2016;Kofuku et al, 2012) or covalent modification (Liu et al, 2012a;Sounier et al, 2015;Bokoch et al, 2010;Eddy et al, 2016) to incorporate isotopic probes into protein from Sf9 cells, and perdeuteration with incorporation of labeled ILV methyl probes is largely intractable for this established eukaryotic system. Another limitation is that purified wild-type GPCRs are typically prone to aggregation and are not highly thermostable, limiting acquisition times and sample concentration.…”

Section: Introductionmentioning

confidence: 99%

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Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

Clark

Dikiy

Chapman

et al. 2018

Biophysical Journal

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GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (A 2A R) that are deuterated apart from 1 H/ 13 C NMR probes at isoleucine d1 methyl groups, which facilitated 1 H/ 13 C methyl TROSY NMR measurements with opposing ligands. Our data indicate that low [Na + ] is required to allow large agonist-induced structural changes in A 2A R, and that patterns of sidechain dynamics substantially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptors, with the inverse agonist suppressing fast ps-ns timescale motions at the G protein binding site. Our approach to GPCR NMR creates a framework for exploring how different regions of a receptor respond to different ligands or signaling proteins through modulation of fast ps-ns sidechain dynamics.

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Section: Resultssupporting

confidence: 58%

Section: Introductionmentioning

confidence: 99%

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

Clark

Dikiy

Chapman

et al. 2018

Biophysical Journal

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“…The rHDLs reportedly provide a lipid environment with more native-like properties, compared with liposomes, in terms of the lateral pressure and curvature profiles because detergent micelles have strong curvature and different lateral pressure profiles from lipid membranes (31). Our NMR analyses of a G protein-coupled receptor (GPCR) and an ion channel in rHDL lipid bilayers revealed that the population and the exchange rates of the conformational equilibrium determine their signal transduction and ion transport activities (32)(33)(34) and that the population of the active conformation of the GPCR in rHDLs correlated better with the signaling levels than that in detergent micelles (32). Therefore, NMR investigations of membrane proteins in the lipid bilayer environments of rHDLs are necessary for accurate measurements of the exchange rates and the populations in conformational equilibrium.…”

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confidence: 99%

Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

Minato

Suzuki

Hara

et al. 2016

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

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Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cationselective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X 4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X 4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s −1 ), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.NMR | membrane proteins | ligand-gated ion channels | insect cell expression system | purinergic receptors I n chemical neurotransmission, various neurotransmitters bind to ligand-gated ion channels expressed in the plasma membrane of postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, leading to changes in membrane potential and the concentration of intracellular ions. Each ligand for a ligand-gated ion channel has a distinct ability to evoke currents (1), and the ligands are classified according to the evoked current level: such as, full agonists, partial agonists, and antagonists. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment (2, 3).Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the open and closed conformations and the distinct conformation of the partial agonist-bound states from the closed and open conformations (4, 5). In the crystal structures of the extracellular region of the AMPA receptor, in which the distances between the two extracellular domains are changed upon agonist binding, the interdomain distances in the partial agonist-bound states correlated with the conductance level, suggesting that the AMPA receptor adopts specific intermediately permeable conformations (4, 6).The P2X receptors are a family of cation channe...

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“…The β 2 -adrenergic receptor (β 2 AR) has been extensively investigated in crystals (4,5), by NMR in solution (6)(7)(8)(9)(10)(11), by bulk fluorescence spectroscopy in solution (12)(13)(14) and in cells (2), by single-molecule fluorescence spectroscopy (15)(16)(17), and by molecular dynamics simulations (18). Despite the availability of highresolution crystal structures of β 2 AR in inactive (4) and active (5) conformations, it remains unknown how ligands regulate transitions between the two states and why β 2 AR exhibits a significant level of ligand-independent, basal signaling activity.…”

mentioning

confidence: 99%

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.

119

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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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Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region

Cited by 184 publications

References 40 publications

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

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

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Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region

Cited by 184 publications

References 40 publications

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

Conductance of P2X 4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

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

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Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

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