Abstract
The thyrotropin (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 HEK-EM293 cells with TSH and measuring cAMP or inositolmonophosphate (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. Persistent IP1 and cAMP signaling were also caused by a small molecule agonist and a thyroid-stimulating antibody. TSH stimulated persistent IP1 production was inhibited by a small molecule inverse agonist and a neutral antagonist 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 Gαq 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 GPCR via the Gq pathway and of two G protein-mediated pathways by any GPCR.
- Thyrotropin/TRH
- Gs family
- Gq/11 family
- cAMP
- IP3/DAG
- Desensitization/uncoupling
- Sequestration/Internalization
- Received March 8, 2011.
- Revision received April 26, 2011.
- Accepted April 26, 2011.
- The American Society for Pharmacology and Experimental Therapeutics