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

Figure 3. Both GB1 and GB2 transmembrane domains (TMDs) are sufficient for the agonist activity of the positive allosteric modulators (PAMs).

(A–F) Intracellular Ca2+ responses mediated by the indicated subunit compositions (pictograms) upon stimulation with rac-BHFF. The inserted graphs correspond to the responses upon stimulation with GABA. Data are normalized by using the response of 100 μM rac-BHFF or 1 mM GABA, for rac-BHFF and GABA treatment, respectively, on wild-type GABAB receptor and shown as means ± SEM of 3–8 biologically independent experiments. The dotted lines in the main and inserted graphs indicate the dose–responses of the wild-type receptor determined in panel (A).

Figure 3—source data 1. Source data for Figure 3, Figure 3—figure supplement 1, Figure 3—figure supplement 2, and Supplementary file 3.
elife-70188-fig3-data1.xlsx (155.6KB, xlsx)

Figure 3.

Figure 3—figure supplement 1. GB1 transmembrane domain (TMD) is required for agonist activity of rac-BHFF.

Figure 3—figure supplement 1.

(A) Quantification of cell surface expression of the indicated HA-tagged GB1 and Flag-tagged GB2 constructs by ELISA in transfected HEK293 cells. Data are normalized by the WT receptor and shown as means ± SEM of 3–18 biologically independent experiments. GB1/2 corresponds to the chimeric subunit made of GB1 ECD and GB2 TMD, and GB2/1 to the chimeric subunit composed of GB2 ECD and GB1 TMD. GB1TM7 stands for the GB1 subunit that is deleted of most of its TMD leaving the seventh helix only. DECD indicates the deletion of the ECD domain. (B–F) Intracellular Ca2+ responses mediated by the indicated constructs upon stimulation with rac-BHFF (B, C, F) or GABA (D, E). Data are normalized by WT response of 100 μM rac-BHFF (B, C, F) or 1 mM GABA (D, E) and shown as means ± SEM of 3–6 biologically independent experiments. Cartoons illustrate the subunit compositions corresponding to the red curves. ASA corresponds to the mutated GB1 subunit in which the endoplasmic reticulum retention signal RSR has been mutated to ASA to allow its efficient trafficking to the cell surface in the absence of GB2. DCRC corresponds to the GB1 mutant in which D6492.59 and R6653.32 in TMD were replaced by cysteines. This mutation is shown as a red cross in panels (E) and (F).
Figure 3—figure supplement 2. GB1 transmembrane domain (TMD) is required for agonist activity of the ago-positive allosteric modulators (ago-PAMs).

Figure 3—figure supplement 2.

(A) BRET ratio changes mediated by the WT (in blue) and indicated constructs upon stimulation with 100 μM rac-BHFF. Data are shown as means ± SEM of 3–7 biologically independent experiments. (B) Inositol-phosphate-1 (IP1) accumulation mediated by the indicated constructs in the presence of 30 μM of the indicated PAM. Data are normalized to the WT response and shown as means ± SEM of three biologically independent experiments. In (A) and (B), 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 and ****p<0.0001.