Figure 3.

Mutant M637W decreases the TSHR activation potency of LMW-Ag. HEK-EM293 cells transiently transfected with wt TSHR or mutant receptors were exposed to increasing concentrations of TSH or the small-molecule ligand LMW-Ag (c2) in the presence of 1 mM IBMX as described in Materials and Methods. After 60 min, the cells were lysed, and cAMP levels were measured by ELISA. Data from 3 independent experiments with duplicate samples are shown. A) In contrast to TSH, LMW-Ag shows a 14-fold decrease in activation of the M637W mutant (wt TSHR EC₅₀: 0.2±0.02 μM; M637W EC₅₀: 2.8±0.6 μM). B) There is no difference between activation of TSHR and the M637C mutant by LMW-Ag (EC₅₀ TSHR: 0.2±0.02 μM; EC₅₀ M637C: 0.4 ±0.3 μM).