Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling

Huang, Shuya Kate; Pandey, Akhilesh; Tran, Duy Phuoc; Villanueva, Nicolas; Kitao, Akio; Sunahara, Roger K.; Sljoka, Adnan; Prosser, R. Scott

doi:10.1016/j.cell.2021.02.041

Cited by 113 publications

(161 citation statements)

References 77 publications

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“…This DXT study confirmed the critical role of the intramolecular ionic lock in switching between active and inactive forms. A recent study using fluorine nuclear magnetic resonance spectroscopy demonstrated adenosine A 2A receptors facilitate at least two distinct active states and three inactive states [ 23 ]. The number of active conformations introduced by the breakage of ionic lock in DRY/AAY still remains unclear.…”

Section: Discussionmentioning

confidence: 99%

“…In this DXT analysis, we mainly focused on the intramolecular dynamics of 5-HT 2A R. However, to understand the total regulation of the receptor, intermolecular association with the effector proteins such as G-proteins and arrestins, and higher ordered movement including receptor dimerization and cell integration should be considered [ 24 ]. Binding of G protein was shown to stabilize an activation intermediate in adenosine A 2A receptors, and Gsαβγ shifts the receptor equilibrium to a predominantly active ensemble [ 23 ]. Furthermore, molecular dynamics simulations have shown that the dynamics of an adenosine A2a receptor vary significantly according to the lipid environment [ 25 ].…”

Section: Discussionmentioning

confidence: 99%

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Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Mio

Fujimura

Ishihara

et al. 2021

IJMS

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Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HT2AR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HT2ARs have been well studied, little has been known about their real-time dynamics. In this study, we analyzed the intramolecular motion of the 5-HT2AR in living cells using the diffracted X-ray tracking (DXT) technique. The DXT is a very precise single-molecular analytical technique, which tracks diffraction spots from the gold nanocrystals labeled on the protein surface. Trajectory analysis provides insight into protein dynamics. The 5-HT2ARs were transiently expressed in HEK 293 cells, and the gold nanocrystals were attached to the N-terminal introduced FLAG-tag via anti-FLAG antibodies. The motions were recorded with a frame rate of 100 μs per frame. A lifetime filtering technique demonstrated that the unliganded receptors contain high mobility population with clockwise twisting. This rotation was, however, abolished by either a full agonist α-methylserotonin or an inverse agonist ketanserin. Mutation analysis revealed that the “ionic lock” between the DRY motif in the third transmembrane segment and a negatively charged residue of the sixth transmembrane segment is essential for the torsional motion at the N-terminus of the receptor.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Mio

Fujimura

Ishihara

et al. 2021

IJMS

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“…This implies that the cell or membrane environment plays a significant role in modulating receptor function. As GPCR reconstitution efforts have improved, it has become possible to directly interrogate the stabilizing effects of phospholipid mixtures over detergent [ 31 , 90 , 91 ]. Of note, agonist affinities and receptor stability increase precipitously in phospholipid.…”

Section: Recapitulating Function In Polymer-stabilized Memteins As Exemplified By a 2a Rmentioning

confidence: 99%

“…Of note, agonist affinities and receptor stability increase precipitously in phospholipid. At the same time, G protein coupling and ligand-dependent modulation of G protein action, as measured through nucleotide exchange or GTP hydrolysis assays, is rarely possible without resorting to reconstituted phospholipid bilayer assemblies [ 91 ]. In contrast, while some of the earlier ligand binding assays in SMALPs clearly demonstrated that a GPCR could be reconstituted [ 36 ], activation and G protein coupling is much more stringent and is expected to require an appropriate lipid interface in addition to an electrostatic surface potential that allows binding an activation of the G protein.…”

Section: Recapitulating Function In Polymer-stabilized Memteins As Exemplified By a 2a Rmentioning

confidence: 99%

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

et al. 2021

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Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins).

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“…While several dozen GPCRs have been reported to be produced in P. pastoris [ 11 ], including adrenergic receptors [ 12 ], muscarinic receptors [ 13 ], dopamine receptors [ 14 ] and opioid receptors [ 15 ], a much smaller number of GPCRs have been successfully produced in P. pastoris for NMR experiments. The most studied example is the human A 2A adenosine receptor [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. More recently, NMR data have been reported for the cannabinoid receptor type 1 [ 24 ] and orexin receptor 2 [ 24 ] proteins expressed in P. pastoris .…”

Section: Introductionmentioning

confidence: 99%

Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions

2021

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G protein-coupled receptors (GPCRs) bind a broad array of extracellular molecules and transmit intracellular signals that initiate physiological responses. The signal transduction functions of GPCRs are inherently related to their structural plasticity, which can be experimentally observed by spectroscopic techniques. Nuclear magnetic resonance (NMR) spectroscopy in particular is an especially advantageous method to study the dynamic behavior of GPCRs. The success of NMR studies critically relies on the production of functional GPCRs containing stable-isotope labeled probes, which remains a challenging endeavor for most human GPCRs. We report a protocol for the production of the human histamine H1 receptor (H1R) in the methylotrophic yeast Pichia pastoris for NMR experiments. Systematic evaluation of multiple expression parameters resulted in a ten-fold increase in the yield of expressed H1R over initial efforts in defined media. The expressed receptor could be purified to homogeneity and was found to respond to the addition of known H1R ligands. Two-dimensional transverse relaxation-optimized spectroscopy (TROSY) NMR spectra of stable-isotope labeled H1R show well-dispersed and resolved signals consistent with a properly folded protein, and 19F-NMR data register a response of the protein to differences in efficacies of bound ligands.

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Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling

Cited by 113 publications

References 77 publications

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions

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