Fig. 4.

Downregulation of TSHR cell surface expression did not affect decreased cAMP production caused by 100 mU/ml bTSH. (A) TSHR cell surface expression in HEK-TSHR cells was measured by FACS after a 60-minute incubation without (basal) or with 100 mU/ml (1.8 µM) bTSH in control cells or cells transfected 2 days before with a plasmid that expresses the dominant negative dynamin 1 K44A mutant and with ARRB2 (β-arrestin 2) siRNA. Expression of dynamin 1 K44A and knockdown of β-arrestin 2 inhibited downregulation of TSHR cell surface expression. The bars represent mean with 95% confidence interval of duplicate or triplicate measurements in four FACS experiments. Statistical significance was determined by unpaired two-tailed t test (***P < 0.001). (B) In parallel to the FACS analysis, cAMP production was measured in control and transfected HEK-TSHR cells. The cells were incubated for 60 minutes with 100 mU/ml TSH in HBSS/HEPES/IBMX. Subsequently, the cells were lysed and cAMP production was analyzed. The bars represent mean with 95% confidence interval. There was no statistically significant difference determined by unpaired two-tailed t test between cells transfected with the dynamin 1 K44A mutant and ARRB2 siRNA and nontreated cells (control). KD, knockdown.