Thyrotropin Receptor Stimulates Internalization-Independent Persistent Phosphoinositide Signaling

Abstract

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 G␣ 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 G␣ q pathway and of two G protein-mediated pathways by any GPCR.

Persistent signaling by thyrotropin‐releasing hormone receptors correlates with G‐protein and receptor levels

“…(2) proposed that in mouse thyrocytes receptor internalization is required to ensure sustained cAMP production and that TSHRs internalized in close association with Gα s subunits and adenylyl cyclase; they recently reported that human embryonic kidney 293 (HEK293) cells do not exhibit persistent signaling by TSHR (8). By contrast, our previous findings (5,7) in HEK‐EM293 cells stably expressing TSHRs have shown that TSHR is capable of signaling persistently via both Gα s and Gα q/11 pathways and that this signaling is independent of internalization.…”

“…The protocol to measure persistent IP1 production, which is defined as continued IP1 production above basal after washout of agonist, was described previously (5,7). Briefly, cells seeded into 24‐well plates at a density of 2.2 × 10 5 cell/well were cultured for 24 h (details for the experiments that required transfections are described above).…”

Kinetics of 5-HT2BReceptor Signaling: Profound Agonist-Dependent Effects on Signaling Onset and Duration

“…In contrast, there are far fewer reports documenting persistent agonist binding and signaling (Calebiro et al, 2009;Mullershausen et al, 2009;Neumann et al, 2010;Boutin et al, 2011;Werthmann et al, 2012). In the present study, the persistent signaling for ergots such as CAB appears to be established in an essentially quantitative manner, since measured potencies did not decrease following extensive compound washout.…”

GPCR signaling along the endocytic pathway

“…further suggested that this specialized form of endosome-based G protein activation directs signaling to qualitatively different downstream effectors [36]. A potential caveat to this interpretation is that a sustained response might occur as a consequence of slow dissociation of TSH from the TSHR irrespective of endocytosis [37]. A follow-up study supported the endosome signaling hypothesis by showing that the duration of the sustained response exceeds the time required for TSH dissociation from TSHRs.…”