Thyrotropin receptor stimulates internalization-independent persistent phosphoinositide signaling.

The thyrotropin [thyroid-stimulating hormone (TSH)] receptor (TSHR) is known to acutely and persistently stimulate cAMP signaling and at higher TSH concentrations to acutely stimulate phosphoinositide signaling. We measured persistent signaling by stimulating TSHR-expressing human embryonic kidney-EM293 cells with TSH and measuring cAMP or inositol monophosphate (IP1) production, a measure of phosphoinositide signaling, 60 min or longer after TSH removal. In contrast to persistent cAMP production, persistent IP1 production increased progressively when TSH exposure was increased from 1 to 30 min, whereas the rates of decay of persistent signaling were similar. A small-molecule agonist and a thyroid-stimulating antibody also caused persistent IP1 and cAMP signaling. A small-molecule inverse agonist and a neutral antagonist inhibited TSH-stimulated persistent IP1 production, whereas the inverse agonist but not the neutral antagonist inhibited persistent cAMP production. As with persistent cAMP production, persistent IP1 production was not affected when TSHR internalization was inhibited or enhanced. Moreover, Alexa546-TSH-activated TSHR internalization was not accompanied by Gα(q) coupling protein internalization. Thus, transient exposure to high concentrations of TSH causes persistent phosphoinositide and cAMP signaling that is not dependent on internalization. To our knowledge, this is the first demonstration of persistent activation by any G protein-coupled receptor (GPCR) via the Gα(q) pathway and of two G protein-mediated pathways by any GPCR.