Figure 5.

Inhibition of TSH signaling by TSHRv1.3 protein. Inhibition of (A) TSH-stimulated cAMP generation and (B) MS-1–induced cAMP generation by increasing concentrations of purified TSHRv1.3 protein was measured using TSHR-Glo cells. For this assay, a fixed concentration of TSH corresponding to 1000 μU/mL was first preincubated with different concentrations of TSHRv1.3 purified protein or control protein, as indicated on the x-axis for 30 minutes at 37°C in a volume of 200 μL. This mixture was then equally divided and added in duplicate to 96-well microtiter plates seeded with 50,000 cells per well of TSHR-Glo cells and incubated overnight at 37°C. Control wells were either stimulated with medium alone or TSH 1000 μU/mL as negative and positive controls. TSHRv1.3 protein could almost totally dampen the induced stimulation of cAMP in this transcriptional-based bioassay. The data in these plots are the mean of two experiments.