Probing GPR126 Conformational Changes

To monitor the extracellular conformational changes of GPR126 deduced by progesterone and 17OHP binding, a FlAsH-BRET assay was performed as previously described (33 (link), 45 (link), 94 (link)). In brief, HEK293 cells were seeded in six-well plate and transfected with GPR126 FlAsH-BRET sensors (S1 to S6, as indicated in SI Appendix, Fig. S5A) or relative mutants based on sensor S4 and incubated for 48 h at 37 °C in 5% CO₂. Then the cells were labeled with 2.5 µM FlAsH EDT2 (Thermo Fisher Scientific) in accordance with the manufacturer’s specification. After that, the cells expressing the labeled GPR126 FlAsH-BRET sensors were seeded into a 96-well plate at a density of 5 × 10⁴ cells per well. The indicated concentrations of progesterone and 17OHP were added into each well together with luciferase substrate coelenterazine-h (5 μM). The BRET signal was calculated as the ratio of FlAsH to Nluc emission. The change in BRET signal due to ligand addition was recorded as ΔBRET.