Fig. 4. Signaling properties of WT and 11 TSHR variants. (A) cAMP production in 293T cells. 293T cells were transfected with the respective TSHR expression vectors (WT or mutant) and incubated with or without 10 U/L bTSH. Intracellular cAMP levels were measured using a cAMP assay kit. Student’s t-test was used to compare the cAMP levels between the WT and each variant. (B) Gq/11 signaling in cells expressing the TSHR variants. Gq/11 signaling in cells expressing the TSHR variants was examined indirectly based on firefly luciferase activity in 293T cells. Cells were treated with or without 100 U/L bTSH, and luciferase activity was measured. Student’s t-test was used to compare luciferase activity between the WT and each variant. Data are representative of three independent experiments (each performed in quadruplicate) with similar results, and the values represent the mean±SE. Note that there are no significant differences in the levels of intracellular cAMP and Gq/11 activity between the variants and the WT in the basal state. *P<0.05, **P<0.01, ***P<0.001.
Abbreviations: WT, wild-type; TSHR, TSH receptor; bTSH, bovine thyroid-stimulating hormone; FT3, free triiodothyronine; FT4, free thyroxine; ns, no significance.

Ann Lab Med 2024;44:343~353 https://doi.org/10.3343/alm.2023.0337