Extended Data Fig. 1 |. Signaling studies for TSHR.

a, b) DNA titrations of TSHR mutants tested in cAMP production assays. Median fluorescence intensity ± SD (n = 3) of anti-FLAG-A647 staining is plotted. Filled in bars represent the DNA concentration used for co-transfection experiments with pGlo cAMP biosensor plasmid. Horizontal red line indicates the mean wild-type (WT) TSHR fluorescence intensity. Flow cytometry experiments were performed in triplicate, with identical gating across all cell lines tested. c) cAMP production of TSHR cysteine mutants comparing the basal level (−) to 100 nM of TSH, M22, or CS-17 for untreated cells (left panel) and cells treated with 500 μM TCEP (right panel). Data points are means ± SD from three biological replicates. d, e) cAMP production curves comparing TSH and M22-mediated activation of WT and C-peptide deleted TSHR constructs. f) cAMP production curves for TSH and TR1402-mediated WT TSHR activation. g) cAMP production curves for TSH-mediated WT and TSHR mutant cell line (Y385F, Y385A) activation. h) cAMP production curves for M22-mediated WT and lipid-displacing TSHR mutant cell line (A644K, A647K) activation. Plotted data points in panels c-g are means of triplicate measurements ± SD from a representative experiment of n = 3 biological replicates.