Volume 24, Issue 3, Pages 360-370 (March 2017) FRET-Based Sensors Unravel Activation and Allosteric Modulation of the GABAB Receptor  Nathalie Lecat-Guillet, Carine Monnier, Xavier Rovira, Julie Kniazeff, Laurent Lamarque, Jurriaan M. Zwier, Eric Trinquet, Jean-Philippe Pin, Philippe Rondard  Cell Chemical Biology  Volume 24, Issue 3, Pages 360-370 (March 2017) DOI: 10.1016/j.chembiol.2017.02.011 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 360-370DOI: (10. 1016/j. chembiol. 2017 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Absence of FRET Modulation between the N-Terminal Ends of GB1 and GB2 in the GABAB Receptor (A) Crystal structures of the VFT dimer for the GABAB and mGlu1 receptors (see indicated PDB numbers) were solved in the presence of antagonist or agonist, revealing the two major conformational states of the ECD. The dimeric VFTs were observed in inactive (left panel) and active (right panel) orientation whether they were bound to antagonist (blue) or to agonist (red). In contrast to the mGluR1, where the two VFTs are in open (left panel) or closed (right panel) state, the GABAB is made of GB1 and GB2 VFTs and only the GB1 VFT binds ligand, either agonist (red) or competitive antagonist (dark blue), while GB2 VFT remains open and unbound by ligand. Comparison of these active and inactive forms shows that the GABAB ECD undergoes a smaller rearrangement to that of mGluR1; the lower lobes of the VFT are distant in the inactive state and in direct contact in the active state. The ECD conformational change can also be measured by the distance between the two N termini in the dimeric VFTs (double-headed arrows). Here the variation is smaller in GABAB relative to mGluR1. The Cα of the first N-terminal residue in the crystal structures is displayed as a black ball. (B) Inter-subunit trFRET signal for STGB1a + STGB2 and STmGluR6. The extracellular N-terminal SNAP-tag (ST) subunits were covalently labeled with SNAP-Lumi4-Tb (donor) and SNAP-Green (acceptor). The trFRET signal was measured in the presence of a saturating concentration of GABAB or mGluR6 competitive antagonist (CGP54626 or LY341495) or agonist (GABA or L-AP4). Data are means ± SEM of three individual experiments, each performed in triplicate. Cell Chemical Biology 2017 24, 360-370DOI: (10.1016/j.chembiol.2017.02.011) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Development of GABAB Receptor trFRET Conformational Sensors (A) Cartoon illustrating the two isoforms of the dimeric GABAB receptor composed of GB1a or GB1b associated with GB2. For orthogonal labeling of the receptor, SNAP-, ACP-, or ACP17-tag was inserted in the three indicated regions of GB1 and GB2. (B and C) trFRET signal of GABAB receptors made of the GB1b and GB2 subunits, after labeling of the indicated tag with a pair of fluorophores. Data are the mean ± SEM of triplicates from representative experiments performed at least three times. Of note, only the inter- and intra-subunit sensors that are well-expressed and well-labeled at the cell surface were shown in this figure. Cell Chemical Biology 2017 24, 360-370DOI: (10.1016/j.chembiol.2017.02.011) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 The Relative Movement of the GABAB ECDs Is Correlated with Receptor Activation (A) Cartoon illustrating the GABAB receptor made of GB1ACP + STGB2 and labeled with CoA-Lumi4-Tb and SNAP-Green, fluorescent donor and acceptor, respectively. The rationale for expecting a high FRET signal in the absence of ligand or in the presence of the antagonist CGP54626 and a low FRET signal in the presence of GABA is illustrated. (B) trFRET signals after cell-surface labeling of GB1ACP and STGB2 with CoA-Lumi4-Tb and SNAP-Green in the presence of a saturating concentration of GABA (injection starting at t = 25 s) and then of CGP54626 (injection at t = 60 s). The trFRET signal is normalized to the maximum signal obtained in the presence of a saturating concentration of antagonist. (C) Inter-subunit trFRET signal measured on GABAB receptor in the presence of the indicated ligands. Of note, these ligands have been co-crystallized with GABAB ECD (Geng et al., 2013). (D) Inter-subunit trFRET signal for the wild-type GABAB receptor, and the ligand binding-deficient GABAB receptor mutant made of GB1bACPS130A + STGB2. Data are mean ± SEM of three individual experiments, each performed in triplicate. (E) 3D model of the GABAB ECD in the inactive and active states where the ACP-tag (yellow) inserted in GB1 was included in the crystal structure of the ECDs. Distances between the Cα of the ACP-tag residue (black), where Lumi4-Tb is attached in GB1, and the Cα of the first N-terminal residue in the crystal structure in GB2 (black), where the SNAP-tag is attached, are indicated. Cell Chemical Biology 2017 24, 360-370DOI: (10.1016/j.chembiol.2017.02.011) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Characterization of Orthosteric Compounds Using the GABAB Conformational Sensor Inter-subunit trFRET signal (A and B), intracellular calcium mobilization (C and D) and BRET ratios between RLuc8-tagged Gαi1 or Gαo and YFP-tagged Gβ1γ2 (E–H) measured on cells co-expressing GB1ACP + STGB2 in the presence of increasing concentrations of the indicated agonists or antagonists. The cells were labeled with CoA-Lumi4-Tb and SNAP-Green (A and B), or not (C–H). Data are means ± SEM of at least five individual experiments (n = 5–10), each performed in triplicate. Cell Chemical Biology 2017 24, 360-370DOI: (10.1016/j.chembiol.2017.02.011) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 G Protein Enhances Agonist Potency on the GABAB Receptor (A) Cartoon illustrating the key intermediate of the GTP-to-GDP exchange, the agonist-receptor-Gempty complex, in which the agonist has a reduced dissociation rate and therefore a higher apparent affinity. (B) Comparison of the signals obtained in the indicated assays on cells co-expressing GB1ACP + STGB2. (C) Cartoon illustrating the mimicking of the agonist-receptor-Gempty complex intermediate state by the Gαo mutant (G203T). (D) Inter-subunit trFRET signal measured on cells co-expressing GB1ACP + STGB2 + wild-type or mutant Gαo. Horizontal and vertical arrows indicate the increase in potency and efficacy, respectively. Data are means ± SEM of at least three individual experiments, each performed in triplicate.
 Cell Chemical Biology 2017 24, 360-370DOI: (10.1016/j.chembiol.2017.02.011) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Different Mechanisms of Action of GABAB PAMs Three PAMs, CGP7930 (A–C), rac-BHFF (D–F), and GS39783 (G–I) were analyzed. Inter-subunit trFRET signal (A, D, and G), BRET ratios of Gαi1 or Gαo (B, E, and H), and intracellular calcium mobilization assay (C, F, and I) measured on cells co-expressing GB1ACP + STGB2 and stimulated with GABA in the absence or presence of the indicated PAM. In trFRET experiments, cells were labeled with CoA-Lumi4Tb and SNAP-Green. Dotted lines show the dose-response of the PAM alone. The arrows indicate the increase of potency and efficacy induced by the indicated PAM. Data are means ± SEM of at least six individual experiments (n = 6–10), each performed in triplicate. Cell Chemical Biology 2017 24, 360-370DOI: (10.1016/j.chembiol.2017.02.011) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Intra-subunit Sensors Reveal TMD Rearrangement in the GABAB Receptor Comparison of the inter-subunit trFRET signal measured on cells co-expressing GB1ACP + STGB2 (A) and STGB1e2ACP + GB2 (B) after labeling with CoA-Lumi4-Tb and SNAP-Green, in the presence of the PAMs alone or of GABA alone. Data are means ± SEM of at least six individual experiments (n = 6–10), each performed in triplicate. Cell Chemical Biology 2017 24, 360-370DOI: (10.1016/j.chembiol.2017.02.011) Copyright © 2017 Elsevier Ltd Terms and Conditions