Endocrine Abstracts

Whereas G-protein coupled receptors (GPCRs) have long been believed to signal via cyclic AMP (cAMP) exclusively at the cell surface, our group has shown that GPCRs also stimulate cAMP production from the cell interior once internalized together with their ligands. This phenomenon, which we originally described for the thyroid stimulating hormone receptor (TSHR) in thyroid follicles, has now also been shown for several other GPCRs. However, the involved subcellular compartment(s) and the mechanisms linking signalling by internalized GPCRs to downstream biological effects are largely unknown. The aim of this study was to follow the trafficking of internalized TSHRs and involved signalling proteins in living primary thyroid cells by highly inclined thin illumination (HILO) microscopy as well as to monitor cAMP production and protein kinase A (PKA) activation by real-time fluorescence resonance energy transfer (FRET) microscopy. Importantly, using a biosensor nanobody that is specific for the active Gs-protein, we were able to directly demonstrate that the internalized TSHR activates an endogenous pool of Gs-protein located on membranes of the Trans Golgi Network (TGN). Direct monitoring of PKA activation with a Golgi-localized FRET sensor (AKAR2) showed a delayed PKA activation (approx. 10 min) after TSH stimulation, which is compatible with the time required for TSH/TSHR complexes to reach the Golgi/TGN. Moreover, our results also indicate that Gs-protein mediated signalling by internalized receptors at the TGN and the resulting PKA activation at a critical position near the nucleus are required for the efficient induction of CREB phosphorylation and gene transcription. These findings provide a mechanism to explain the biological relevance of GPCR signalling at intracellular sites, which might help explaining the pathogenesis of endocrine disorders such as Graves’ disease and might pave the way to innovative pharmacological therapies.