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Proc Natl Acad Sci U S A. 2019 May 14;116(20):10150-10155. doi: 10.1073/pnas.1900261116. Epub 2019 Apr 25.

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

Author information

1
Institute of Pharmacology and Toxicology, University of Würzburg, 97078 Würzburg, Germany.
2
Receptor Signaling Group, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
3
Institute for Physiology II, Jena University Hospital, Friedrich Schiller University Jena, 07740 Jena, Germany.
4
Institute for Physiology II, Jena University Hospital, Friedrich Schiller University Jena, 07740 Jena, Germany klaus.benndorf@med.uni-jena.de lohse@toxi.uni-wuerzburg.de.
5
Institute of Pharmacology and Toxicology, University of Würzburg, 97078 Würzburg, Germany; klaus.benndorf@med.uni-jena.de lohse@toxi.uni-wuerzburg.de.

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; confocal patch-clamp fluorometry; kinetics; metabotropic glutamate receptors; photouncaging

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