Thyrotropin Causes Dose-dependent Biphasic Regulation of cAMP Production Mediated by G_s and G_i/o Proteins

The thyrotropin (TSH) receptor (TSHR) signals via G proteins of all four classes and β-arrestin 1. Stimulation of TSHR leads to increasing cAMP production that has been reported as a monotonic dose-response curve that plateaus at high TSH doses. In HEK 293 cells overexpressing TSHRs (HEK-TSHR cells), we found that TSHR activation exhibits an "inverted U-shaped dose-response curve" with increasing cAMP production at low doses of TSH and decreased cAMP production at high doses (>1 mU/ml). Since protein kinase A inhibition by H-89 and knockdown of β-arrestin 1 or β-arrestin 2 did not affect the decreased cAMP production at high TSH doses, we studied the roles of TSHR downregulation and of G_i/G_o proteins. A high TSH dose (100 mU/ml) caused a 33% decrease in cell-surface TSHR. However, because inhibiting TSHR downregulation with combined expression of a dominant negative dynamin 1 and β-arrestin 2 knockdown had no effect, we concluded that downregulation is not involved in the biphasic cAMP response. Pertussis toxin, which inhibits activation of G_i/G_o, abolished the biphasic response with no statistically significant difference in cAMP levels at 1 and 100 mU/ml TSH. Concordantly, co-knockdown of G_i/G_o proteins increased cAMP levels stimulated by 100 mU/ml TSH from 55% to 73% of the peak level. These data show that biphasic regulation of cAMP production is mediated by G_s and G_i/G_o at low and high TSH doses, respectively, which may represent a mechanism to prevent overstimulation in TSHR-expressing cells. SIGNIFICANCE STATEMENT: We demonstrate biphasic regulation of TSH-mediated cAMP production involving coupling of the TSH receptor (TSHR) to Gs at low TSH doses and to G_i/o at high TSH doses. We suggest that this biphasic cAMP response allows the TSHR to mediate responses at lower levels of TSH and that decreased cAMP production at high doses may represent a mechanism to prevent overstimulation of TSHR-expressing cells. This mechanism could prevent chronic stimulation of thyroid gland function.