Fig. 7.

DREAM-derived peptides enhance TSHR activity. A, Increased cAMP levels in TSHR-10,000 cells after transfection with DREAM or DREAM fragments, schematically represented next to each bar. Data are mean ± sem. Significant differences from control (pcDNA3) by unpaired Student’s t test are indicated: **, P < 0.01; ***, P < 0.001. The sequence of three overlapping peptides (P1, P2, and P3) that cover amino acids 43-90 in DREAM is also shown. B, TSHR-10,000 cells were incubated with DREAM-derived peptides P1, P2, and P3 or a nonrelated peptide as negative control. Cells transfected with DREAM (DR) were included as positive control. Western blot with TSHR, phospho-CREB (P-CREB), and total CREB is shown. C–E, Effect on cAMP levels in TSHR-10,000 cells (C), thyroid follicular carcinoma ML-1 cells (D), and papillary carcinoma NPA cells (E) after incubation with DREAM-derived peptides (2 μm), a control nonrelated peptide (2 μm) or TSH (0.5 mU/ml, 10 min) used as a positive control. Data are mean ± sem. Significant differences from control by unpaired Student’s t test are indicated: ***, P < 0.001. F, Coimmunoprecipitation of TSHR and Gαs with an anti-TSHR antibody using whole-cell extracts from TSHR-10,000 cells transfected with DREAM (DR, upper panel), the P1 peptide (lower panel), and the corresponding controls, empty vector, or nonrelated peptide, respectively. Coimmunoprecipitation with a nonrelated antibody (anti-HA) was used as a negative control. Treatment with TSH (0.5 mU/ml, 10 min) was used as a control. Input corresponds to 1% of total extracts.