Extended Data Fig. 3: Functional characterization of MT1-mutants.
From: Cryo-EM structure of the human MT1–Gi signaling complex
a–c, NanoBiT-G-protein dissociation assays for the constructs used in the cryo-EM analysis and the MT1 mutants. Concentration–response curves for melatonin-dependent G-protein dissociation signals for MT1. To match the expression of MT1-WT to that of mutants with lower expression, 1:5 volume [WT (1:5)] or 1:10 volume [WT (1:10)] MT1-WT plasmid was used. Asterisks denote an increased volume (2.5-fold) of plasmid transfection. Symbols and error bars represent mean and s.e.m. of the indicated independent numbers of experiments (n = 3–5; see Extended Data Table S1). Note that, in most data points, error bars are smaller than the size of the symbols and thus are not visualized in the panels. The results for the construct used in the cryo-EM analysis (a), the F1965.47A residue that is important in MT1-activation (b), and the mutants to test Gi-selectivity (c) are shown. d, Cell surface expression levels of the MT1-constructs. Cells transiently expressing the FLAG epitope-tagged MT1-constructs were labeled with an anti-FLAG tag antibody, and then with an Alexa 488-conjugated secondary antibody. The fluorescent signals from individual cells were measured by a flow cytometer (See Methods). Data from each experiment are shown as dots with the mean value presented as a bar (n = 3–9). For the multiple comparison analysis, two-way ANOVA followed by the Dunnett’s test (WT (1:1) vs. mutant) was performed: ns, not significantly different, *, P < 0.05, *** P < 0.001. Source data for (a–d) are available online.
