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Stepwise activation of a class C GPCR begins with millisecond dimer rearrangement

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Item Type:Article
Title:Stepwise activation of a class C GPCR begins with millisecond dimer rearrangement
Creators Name:Grushevskyi, E.O. and Kukaj, T. and Schmauder, R. and Bock, A. and Zabel, U. and Schwabe, T. and Benndorf, K. and Lohse, M.J.
Abstract:G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within ~1 ms, while all other receptors are thought to become activated within ~50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ(1) ~ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ(2) ~ 20 ms). Receptor deactivation occurred with time constants of ~40 and ~900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.
Keywords:G Protein-Coupled Receptors, Kinetics, Confocal Patch-Clamp Fluorometry, Metabotropic Glutamate Receptors, Photouncaging, Animals, Xenopus laevis, Xenopus oocytes
Source:Proceedings of the National Academy of Sciences of the United States of America
ISSN:0027-8424
Publisher:National Academy of Sciences (U.S.A.)
Volume:116
Number:20
Page Range:10150-10155
Date:14 May 2019
Official Publication:https://doi.org/10.1073/pnas.1900261116
PubMed:View item in PubMed

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