Extended Data Fig. 8: Effects of a PAM on the orthosteric ligand binding in the site-1 and -2 mutants of GABAB.
From: Structural basis of the activation of a metabotropic GABA receptor
a, Displacement of the CGP54626-DY647 by GABA in intact cells expressing the wild-type GABAB receptor in the absence (blue) or presence of 5 μM (red), 10 μM (orange) or 20 μM (sand) GS39783. b, Cell-surface expression of site-1 and -2 mutants, measured by cotransfecting Halo–GB1 with GB2 and recording fluorescence emission of Halo–Lumi4-Tb. Data are normalized by the wild-type expression and shown as mean ± s.d. The numbers of biologically independent experiments are shown in parentheses. c, pKi values for GABA were determined from displacement of CGP54626-DY647 binding in intact cells expressing the indicated subunit combinations in the absence or presence of 1 μM or 5 μM GS39783. Values are mean ± s.e.m. of 4 (10 for wild type) biologically independent experiments. Data are analysed using one-way ANOVA with Dunnett’s multiple comparison test to determine significance (compared with no GS39783 for the same combined subunits), with ****P ≤ 0.0001, ***P ≤ 0.001, *P ≤ 0.01 and NS (not significant) P > 0.05. For 1 μM (5 μM) GS39783, P = 0.0030 (0.0045) for GB2(R556A), 0.0205 (0.0157) for GB2(H647A), 0.0084 (0.0009) for GB2(L657A), 0.0436 (0.0003) for GB2(F711A), 0.0013 (0.0053) for GB2(R714A), 0.0373 (0.0056) for GB2(Q720A), and 0.0735 (0.0019) for GB2(SFR-AAA). GB2(SFR-AAA) refers to a triple mutant (S710A/F711A/R714A) of GB2. d, e, Displacement of the CGP54626-DY647 by GABA in intact cells expressing the indicated mutants in site 1 (d) and site 2 (e), in the absence or presence of the indicated concentrations of GS39783 as in a. Data are normalized by the signal in absence of GS39783 and shown as mean ± s.e.m. of three biologically independent experiments.
