Figure 1

Functional characterization of WT and mutant TSHR. Cell surface expression (A) and Gs cAMP accumulation (B) were determined in COS-7 cells; Gq/₁₁ IP3 accumulation (C) was determined in HEK-293 cells, both expressing WT and mutant (I486N) TSHRs. The cells were transiently transfected with WT TSHR and mutant TSHR-I486N DNA. A: Cell surface expression was measured 72 h after transfection. The cells were fixed, and immunological determination of the N-terminal HA was performed. Results (means ± SD) are expressed as percentage of TSHR-WT (100%). B: Activation of the Gs/adenylyl cyclase pathway was determined 48 h after transfection. Cells were stimulated with bTSH, and intracellular cAMP accumulation was measured. Basal activity is indicated in white bars; activity after stimulation with 100 mU bTSH/ml is indicated in black bars. C: Gq/11 activation was reported 48 h after COS-7 co-transfection with TSHR DNA and a reporter construct containing a response element and the firefly luciferase gene under the control of nuclear factor of activated T-cells (transcription factor). The cells were stimulated with bTSH and lysed, and luciferase activity expression was measured. Basal activity is indicated in white bars, activity after stimulation with 100 mU bTSH/ml in black bars. The figure depicts means ± SD out of duplex or tripletts (n= 2-3). RLU, Relative light units. (*** means p value <0.001). The statistical analysis was made by using a nonparametric One-way-Anova and a tukey-test for column statistic of GraphPad Prism Version 4.03.