Extended Data Fig. 1: Purification of the GPR156-Go complex.
a–c, Size-exclusion chromatography profile (a), SDS-PAGE gel (b), and negative-staining microscopy analysis (c) of the purified GPR156-Go complex. Purification experiments of the GPR156-Go complex in (b) were performed independently at least three times. Negative-staining microscopy analysis (c) was performed once. d, Representative cryo-EM micrograph of the GPR156-Go complex. The cryo-EM data collection of GPR156-Go was performed once. e, 2D class averages of the Go-free GPR156. f, 2D class averages of the GPR156-Go complex. g, Comparison of constitutive activities of GPR156 and GABAB in G-protein activation, measured by BRET2 assay. The activity of GABAB was observed in the absence or presence of 100 µM GABA. Data were mean ± SEM from three independent experiments, performed in technical triplicate. Statistical differences in ΔBRET were analyzed by two-way ANOVA with Dunnett’s post hoc test. h, cAMP inhibition assay for GPR156 C-terminal truncation. Mock transfected with an empty vector was used as a negative control. cAMP production was normalized to a percent of WT activity. i, Surface expression levels of WT and GPR156 mutants in the cAMP assay, measured by ELISA. Surface expression levels of GPR156 mutants were normalized to a percent of WT surface expression level. Data in h and i were mean ± SEM from at least three independent experiments, performed in technical triplicate. Statistical differences were analyzed by one-way ANOVA with Dunnett’s post hoc test, compared to WT (NS, not significant; ***P < 0.001; ****P < 0.0001). j, k, A model of GPR156 predicted from AlphaFold2 was used for initial docking and model building. The N-terminal (1 to 39) and the C-terminal (336 to 814) regions are disordered and omitted in the figure. The model is colored according to the predicted Local Distance Difference Test (pLDDT) score (j). The Predicted-Alignment-Error (PAE) plot of the GPR156 model (k).
