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Conformational dynamics of activation for the pentameric complex of dimeric G protein-coupled receptor and heterotrimeric G protein.

ABSTRACT: Photoactivation of rhodopsin (Rho), a G protein-coupled receptor, causes conformational changes that provide a specific binding site for the rod G protein, G(t). In this work we employed structural mass spectrometry techniques to elucidate the structural changes accompanying transition of ground state Rho to photoactivated Rho (Rho(?)) and in the pentameric complex between dimeric Rho(?) and heterotrimeric G(t). Observed differences in hydroxyl radical labeling and deuterium uptake between Rho(?) and the (Rho(?))(2)-G(t) complex suggest that photoactivation causes structural relaxation of Rho following its initial tightening upon G(t) coupling. In contrast, nucleotide-free G(t) in the complex is significantly more accessible to deuterium uptake allowing it to accept GTP and mediating complex dissociation. Thus, we provide direct evidence that in the critical step of signal amplification, Rho(?) and G(t) exhibit dissimilar conformational changes when they are coupled in the (Rho(?))(2)-G(t) complex.

SUBMITTER: Orban T

PROVIDER: S-EPMC3351692 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Conformational dynamics of activation for the pentameric complex of dimeric G protein-coupled receptor and heterotrimeric G protein.

Orban Tivadar T Jastrzebska Beata B Gupta Sayan S Wang Benlian B Miyagi Masaru M Chance Mark R MR Palczewski Krzysztof K

Structure (London, England : 1993) 20120501 5

Photoactivation of rhodopsin (Rho), a G protein-coupled receptor, causes conformational changes that provide a specific binding site for the rod G protein, G(t). In this work we employed structural mass spectrometry techniques to elucidate the structural changes accompanying transition of ground state Rho to photoactivated Rho (Rho(∗)) and in the pentameric complex between dimeric Rho(∗) and heterotrimeric G(t). Observed differences in hydroxyl radical labeling and deuterium uptake between Rho(∗ ...[more]

PMID: 22579250

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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

Conformational dynamics of activation for the pentameric complex of dimeric G protein-coupled receptor and heterotrimeric G protein.

Publications

Conformational dynamics of activation for the pentameric complex of dimeric G protein-coupled receptor and heterotrimeric G protein.

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