Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 1. Cryo-EM structures of 5-HT4, 5-HT6, and 5-HT7 signaling complexes (A) Serotonin signaling through different subfamilies serotonin receptors. (B–E) The cryo-EM density map (left) and model (right) of the serotonin bound complexes of 5-HT4- Gs (B), 5-HT4-Gi (C), 5-HT6-Gs (D), and the 5-CT bound complex of 5-HT7-Gs (E). See also Figures S1 and S2 and Table S1.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Cryo-EM Sample Prep

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 2. Molecular recognition of 5-HT4, 5-HT6, and 5-HT7 receptors (A–D) Conformation of the ligand-binding pocket in the serotonin-bound 5-HT4-Gs complex (A). Schematic representation of 5-HT-binding interactions. Hydrogen bonds are shown as black dashed lines. Hydrophobic contacts and amino acids are shown in green (B). Ligand-binding pocket shown as surface (C). 5-HT-induced b-arrestin2 recruitment assay for 5-HT4 mutants using NanoBiT. All data are presented as mean values ± SEM with a minimum of four technical replicates and n = 3 biological replicates. See Figure S3 for dose response curves (D). (E–H) Conformation of the ligand-binding pocket in the serotonin-bound 5-HT6-Gs complex (E). Schematic representation of 5-HT-binding interactions. Hydrogen bonds are shown as black dashed lines. Hydrophobic contacts and amino acids are shown in green (F). Ligand-binding pocket shown as surfaces (G). 5-HT- induced b-arrestin2 recruitment assay for 5-HT4 mutants using NanoBiT. All data are presented as mean values ± SEM with a minimum of four technical replicates and n = 3 biological replicates. See Figure S3 for dose response curves (H). (I–L) Conformation of the ligand-binding pocket in the 5-CT bound-5-HT7-Gs complex (I). Schematic representation of 5-HT-binding interactions. Hydrogen bonds are shown as black dashed lines. Hydrophobic contacts and amino acids are shown in green (J). Ligand-binding pocket shown as surfaces (K). Gs- cAMP accumulation results of 5-HT7 mutants activated by 5-CT. All data are presented as mean values ± SEM with a minimum of four technical replicates and n = 3 biological replicates. See Figure S3 for dose response curves (L). * The activation of the mutant is too low to determine EC50, which the corresponding DpEC50 shown as the LogEC50 value of the wild-type receptor. See also Figure S3.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Ligand Binding Assay, Binding Assay, Activation Assay, Mutagenesis

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 3. Comparisons of serotonin binding poses and 5-CT selectivity in different serotonin receptors (A–C) Comparison of ligand recognition between 5-HT4- and 5-HT6-binding serotonin (A). Comparison of ligand recognition between 5-HT4 and 5-HT1A binding serotonin (B). Comparison of ligand recognition between 5-HT4 and 5-HT1D binding serotonin (C). (D–I) Comparison of residues in 5-HT7 recognizing 5-CT with the corresponding residues among other serotonin receptors. PDB code: 5-HT1A, 7E2Y; 5-HT1D, 7E32; 5-HT1E, 7E33; 5-HT2A, 6WHA. See also Figures S3 and S4.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Binding Assay, Comparison

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 4. G protein coupling of 5-HT4, 5-HT6, and 5-HT7 receptors (A, D, G) Interactions between 5-HT4 and Gas subunits. Electronic interaction between the 5-HT4 and Gas protein binding interface (A). Residues model of interaction is shown in (D) and (G). (B, E, H) Interactions between 5-HT6 and Gas subunits. Electronic interaction between the 5-HT6 and Gas protein binding interface (B). Residues model of interaction is shown in (E) and (H). (C, F, I) Interactions between 5-HT7 and Gas subunits. Electronic interaction between the 5-HT7 and Gas protein binding interface (C). Residues model of interaction is shown in (F) and (I). (J) Comparison of the Ga conformation among the structures of Gs-coupled 5-HT receptors. See also Figures S4 and S6.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Protein Binding, Comparison

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 5. Comparison of the 5-HT4-GS com- plex with the 5-HT4-Gi complex (A) Structural alignment of the 5-HT4-Gs and 5-HT4-Gi complexes. (B) Superposition of TM5 and the Gas and Gai subunits. (C) Differences in the a5 helix of Gas and Gai subunits. (D) Differences in the aN helix of Gas and Gai subunits. (E) The relative tilt/rotation angle between the C-terminal a5 helices in the Gs- and Gi-coupled complexes of 5-HT4. (F) Differences in angle and position of the whole Ga-subunit (except the N terminus helix) between the Gs- andGi/o-coupled5-HT4.See also Figures S4 and S5 and Table S4.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Comparison

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 7. Differences between Gs- and Gi-coupled serotonin receptors (A–C) Receptor (5-HT4, A; 5-HT6, B; 5-HT7, C) interactions with the a5 terminus of Gas subunit. (D–F) Receptor (5-HT1A, D; 5-HT1D, E; 5-HT1E, F) interactions with the a5 terminus of Gai subunit. See also Figures S5 and S7 and Table S4.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: