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. 2021 Dec 6;10:e70188. doi: 10.7554/eLife.70188

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© 2021, Liu et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 5. — (A) Cartoon highlighting a possible second binding site (dotted oval) for the PAMs in the ancestral ligand binding pocket of the GB2 TMD. In the structure of the inactive state, this pocket is occupied by one molecule of phospholipid (shown as yellow sticks). The residues (α-carbon) of this phospholipid binding pocket (green) and the residues underneath (red) were changed to evaluate their importance in the agonist activity of rac-BHFF. The highly conserved residues L^3.36 and Y^5.44 were mutated into Ala; G^6.53 conserved in GB2 were changed to Thr conserved at this position in GB1, or Phe conserved at this position for other class C GPCRs such as mGlu and CaSR; in Mut 8, the non-conserved residues ^7.26NVQ^7.28 were mutated in their equivalent in GB2 Drosophila; V^7.35 conserved in GB2 (Val or Phe) was changed to Phe. (B) Intracellular Ca²⁺ responses mediated by the indicated GB2 mutants (M1 to M16) coexpressed with the wild-type GB1 subunit upon stimulation with 30 μM rac-BHFF or 1 mM GABA. Data are normalized by wild-type response and expressed as means ± SEM of three biologically independent experiments. (C) Intracellular Ca²⁺ responses mediated by the indicated constructs upon stimulation with rac-BHFF in the presence of EC₂₀ GABA of each combination. Data are normalized by wild-type response of 100 μM rac-BHFF + EC₂₀ GABA and shown as means ± SEM. (D) Basal inositol-phosphate-1 (IP₁) accumulation mediated by the indicated constructs. Data are normalized by the response of the wild-type and shown as means ± SEM of five biologically independent experiments. Data are analyzed using one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT) with ***p<0.0005, ****p<0.0001. (E) Correlation between the GABA_B constitutive activity measured using the G_o protein BRET sensor and the rac-BHFF agonist effect for the WT GB1 subunit coexpressed with the indicated GB2 mutants. Data are normalized by the response of the wild-type and shown as means ± SEM. (F) Basal IP₁ accumulation mediated by the indicated constructs, including the genetic mutation A567T identified in human GB2 that is equivalent to the mutation A566T in rat. Data are normalized by the response of the WT and shown as means ± SEM of 3–4 biologically independent experiments. Data are analyzed using an unpaired t-test for human, and one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT) for the rat, with *p<0.05, ***p<0.0005, ****p<0.0001. (G) Top view of the sodium binding pocket within the structure of human A_2A adenosine receptor (PDB 4EIY) where the three residues important for Na⁺ interactions are highlighted (Cα in red), the equivalent residues identified in human GB2 TMD (PDB 6UO8), and human mGluR5 TMD (PDB 4OO9); the X.50 numbers shown for A_2AAR are equivalent to the numbers in mGluR5 on the basis of X.50 residues defined in Doré et al., 2014; TM2, TM3, and TM7 that contain the residues involving the identified region are highlighted in cyan. (H) Basal IP₁ accumulation mediated by the indicated WT and mutated mGlu5 receptors in the presence of the co-transfected glutamate transporter EAAT3. Data are normalized by the response of the WT and shown as means ± SEM of four biologically independent experiments. Data are analyzed using one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT), with **p<0.005. For clarity, the residue numbers for GB2 subunit are based on the sequence of rat GB2. Negative controls (Ctrl) are HEK293 cells co-transfected with the empty vector and Gαqi₉ cDNA (B), or Gαo-Rluc and Gβ₁γ₂-Venus cDNAs in the absence of receptor (E).

Figure 5—source data 1. Source data for Figure 5B–F and H, Figure 5—figure supplement 2, Figure 5—figure supplement 3, Figure 5—figure supplement 4A and B, Figure 5—figure supplement 4E and F, and Supplementary file 2.

elife-70188-fig5-data1.xlsx^{(248.5KB, xlsx)}

Figure 5—figure supplement 1. — (A) Cartoon highlighting a possible second binding site (dotted oval) for the PAMs in the ancestral ligand binding pocket of the GB2 TMD. In the structure of the inactive state, this pocket is occupied by one molecule of phospholipid (shown as yellow sticks). The residues (α-carbon) of this phospholipid binding pocket (green) and the residues underneath (red) were changed to evaluate their importance in the agonist activity of rac-BHFF. The highly conserved residues L^3.36 and Y^5.44 were mutated into Ala; G^6.53 conserved in GB2 were changed to Thr conserved at this position in GB1, or Phe conserved at this position for other class C GPCRs such as mGlu and CaSR; in Mut 8, the non-conserved residues ^7.26NVQ^7.28 were mutated in their equivalent in GB2 Drosophila; V^7.35 conserved in GB2 (Val or Phe) was changed to Phe. (B) Intracellular Ca²⁺ responses mediated by the indicated GB2 mutants (M1 to M16) coexpressed with the wild-type GB1 subunit upon stimulation with 30 μM rac-BHFF or 1 mM GABA. Data are normalized by wild-type response and expressed as means ± SEM of three biologically independent experiments. (C) Intracellular Ca²⁺ responses mediated by the indicated constructs upon stimulation with rac-BHFF in the presence of EC₂₀ GABA of each combination. Data are normalized by wild-type response of 100 μM rac-BHFF + EC₂₀ GABA and shown as means ± SEM. (D) Basal inositol-phosphate-1 (IP₁) accumulation mediated by the indicated constructs. Data are normalized by the response of the wild-type and shown as means ± SEM of five biologically independent experiments. Data are analyzed using one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT) with ***p<0.0005, ****p<0.0001. (E) Correlation between the GABA_B constitutive activity measured using the G_o protein BRET sensor and the rac-BHFF agonist effect for the WT GB1 subunit coexpressed with the indicated GB2 mutants. Data are normalized by the response of the wild-type and shown as means ± SEM. (F) Basal IP₁ accumulation mediated by the indicated constructs, including the genetic mutation A567T identified in human GB2 that is equivalent to the mutation A566T in rat. Data are normalized by the response of the WT and shown as means ± SEM of 3–4 biologically independent experiments. Data are analyzed using an unpaired t-test for human, and one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT) for the rat, with *p<0.05, ***p<0.0005, ****p<0.0001. (G) Top view of the sodium binding pocket within the structure of human A_2A adenosine receptor (PDB 4EIY) where the three residues important for Na⁺ interactions are highlighted (Cα in red), the equivalent residues identified in human GB2 TMD (PDB 6UO8), and human mGluR5 TMD (PDB 4OO9); the X.50 numbers shown for A_2AAR are equivalent to the numbers in mGluR5 on the basis of X.50 residues defined in Doré et al., 2014; TM2, TM3, and TM7 that contain the residues involving the identified region are highlighted in cyan. (H) Basal IP₁ accumulation mediated by the indicated WT and mutated mGlu5 receptors in the presence of the co-transfected glutamate transporter EAAT3. Data are normalized by the response of the WT and shown as means ± SEM of four biologically independent experiments. Data are analyzed using one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT), with **p<0.005. For clarity, the residue numbers for GB2 subunit are based on the sequence of rat GB2. Negative controls (Ctrl) are HEK293 cells co-transfected with the empty vector and Gαqi₉ cDNA (B), or Gαo-Rluc and Gβ₁γ₂-Venus cDNAs in the absence of receptor (E).

Figure 5—source data 1. Source data for Figure 5B–F and H, Figure 5—figure supplement 2, Figure 5—figure supplement 3, Figure 5—figure supplement 4A and B, Figure 5—figure supplement 4E and F, and Supplementary file 2.

elife-70188-fig5-data1.xlsx^{(248.5KB, xlsx)}