Figure 8.

Inverse agonism of Org 274179-0 at constitutively active mutant and wild-type (WT) TSH receptors. (A) DNA dose-dependent increase in basal cAMP synthesis in CHO cells transfected with WT and constitutively active mutant TSH receptors. CHO cells were transiently transfected with 10, 80 and 240 ng receptor cDNA in pKCR per 25 000 cells per well of 96 well plates and incubated for 60 min at 37°C. cAMP was measured using the AlphaScreen kit. (B) CHO cells were transiently transfected with 100 ng receptor cDNA per 15 000 cells and incubated with the indicated concentrations of Org 274179-0 for 60 min at 37°C. Basal cAMP concentrations of the I568T, D619G, A623V, T632I and D633E TSHR mutants were increased 3.0-, 2.3-, 2.6-, 2.9- and 2.0–fold over the basal activity of the WT TSH receptor, respectively. The maximum cAMP levels induced by 316 nM TSH at the mutant and WT TSH receptors were between 100 and 128 nM. Basal cAMP levels of the mutant TSH receptors were between 52% and 74% of the maximum cAMP levels induced by TSH (data not shown). Data are the mean ± SEM of a single experiment, done in quadruplicate, and representative of two independent experiments.