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Single-molecule observation of the ligand-induced population shift of rhodopsin, a G-protein-coupled receptor.

ABSTRACT: Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.

SUBMITTER: Maeda R

PROVIDER: S-EPMC3945020 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Single-molecule observation of the ligand-induced population shift of rhodopsin, a G-protein-coupled receptor.

Maeda Ryo R Hiroshima Michio M Yamashita Takahiro T Wada Akimori A Nishimura Shoko S Sako Yasushi Y Shichida Yoshinori Y Imamoto Yasushi Y

Biophysical journal 20140201 4

Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single- ...[more]

PMID: 24559994

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Project description:BackgroundArabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor-like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction.MethodsTherefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy.ResultsThe LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants.ConclusionsA temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP. Finally, the G protein complex becomes dissociated thus AGB1 interacts with its effector proteins leading to changes in reactive oxygen species and calcium.

Dataset Information

Single-molecule observation of the ligand-induced population shift of rhodopsin, a G-protein-coupled receptor.

Publications

Single-molecule observation of the ligand-induced population shift of rhodopsin, a G-protein-coupled receptor.

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