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. 2018 Jul 10;8:10414. doi: 10.1038/s41598-018-28682-6

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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mGlu₂ dimers are in close physical proximity in the active state. LRET between mGlu₂ dimers with a cysteine introduced in TM4 (V699C) that can form disulfide bonds between dimers upon incubation with the oxidizing agent copper phenanthroline (CuP) when the receptor is in the active state. (a) Schematic drawing of the experimental set up. A quality control system based on modified GABA_B C-terminal domains was used to ensure that each mGlu₂ dimer only contained one SNAP-tag (1ST-mGlu₂). LRET was measured between SNAP-Lumi4-Tb (donor) and SNAP-Red (acceptor) covalently attached to the SNAP-tags. (b) Scatter plot of the TR-FRET intensity in the low expression range (dashed lines in Supplementary Fig. S6). In this interval the TR-FRET intensity for 1ST-mGlu₂-V699C incubated with CuP in presence of agonist and PAM (10 nM LY379268 and 3 μM BINA) is significantly higher than when the same receptor mutant is incubated with CuP in presence of antagonist (400 nM LY341495) or the negative control 1ST-mGlu₂ incubated with CuP in presence of agonist and PAM (p < 0.0001, one-way ANOVA, Bonferroni’s multiple comparisons test). Data are from 4 (1ST-mGlu₂-V699C+ agonist and PAM) or 2 (controls) independent experiments. (c) Representative donor, acceptor and TR-FRET images of 1ST-mGlu₂-V699C incubated with CuP in presence of either agonist and PAM or antagonist, and 1ST-mGlu₂ incubated with CuP in presence of agonist and PAM.