Fig. 3: Evolution of PTH1R in mammalian cells yields signaling-active receptor variants with increased ligand affinity.

a Comparison of the populations after selection with PTH’(1–34)-HL647 (left panel) or M-PTH(1–14)-HL647 (right panel) to wild-type PTH1R: sort 1 (red line), sort 2 (blue line), sort 3a (black line), sort 3b (green line, repetition of 3a), negative control (dark gray shaded). b Expression analysis of 43 evolved PTH1R variants assessed in live HEK293T cells by flow cytometry analysis with saturating concentrations of PTH’(1–34)-HL647. Expression levels are relative to wild-type receptor expression and are given as mean values ± s.e.m. of 2–3 independent experiments (Supplementary Table 3). c cAMP accumulation of 43 evolved PTH1R variants after stimulation with 1 µM PTH(1–34). Data represent maximal cAMP concentrations relative to PTH1R. Bars represent mean values ± s.e.m. of 3–6 independent experiments performed in duplicates (Supplementary Table 4). d Ligand affinities of of 43 evolved PTH1R variants in comparison to PTH1R. IC₅₀ values were derived from whole-cell ligand competition-binding experiments with M-PTH(1–14) or PTH(1–34). Bars represent the mean change ± s.e.m. in calculated affinity (∆pIC₅₀) for each mutant compared with wild-type receptor from 2–8 independent experiments performed in duplicates (Supplementary Table 3). b–d Ligands used for selection are indicated below the bar plots. Source data are provided as a Source Data file.