Endocrine Abstracts

Whereas G-protein coupled receptors (GPCRs) have been long believed to signal through cyclic AMP exclusively at cell surface, our group has previously shown that GPCRs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent cAMP production. This phenomenon, which we originally described for the thyroid stimulating hormone receptor (TSHR) in thyroid cells, has been observed also for other GPCRs. However, the intracellular compartment(s) responsible for such persistent signaling and its consequences on downstream effectors were insufficiently characterized. The aim of this study was to follow by live-cell imaging the trafficking of internalized TSHRs and other involved signaling proteins as well as to understand the consequences of signaling by internalized TSHRs on the downstream activation of protein kinase A (PKA). PKA activity was measured in real-time in living thyroid cells using a fluorescence resonance energy transfer (FRET) sensor (AKAR2). Our results suggest that TSH co-internalizes with its receptor and that the internalized TSH/TSHR complexes traffic retrogradely to the trans-Golgi network (TGN). We provide evidence that TSH/TSHR complexes meet an intracellular pool of Gαs in the TGN and activate it there, as visualized in real-time using a nanobody that binds selectively to the active Gs protein. Acute Brefeldin A-induced Golgi collapse hinders the retrograde trafficking of TSH/TSHR complexes, leading to reduced cAMP production. Direct monitoring of PKA activation in the TGN with a localized AKAR2 sensor shows a delayed PKA activation after TSH stimulation (~10 min), which is compatible with the time required for TSH/TSHR complexes to reach the TGN. These data provide evidence that internalized TSH/TSHR complexes meet and activate G-proteins at the TGN, leading to a local activation of PKA. These findings suggest unexpected functions for receptor internalization, with major pathophysiological and pharmacological implications.