pbio-1000172-g006: Role of PDEs on cAMP signal irreversibility.(A) Thyroid follicles isolated from CAG-Epac1-camps mice were stimulated with TSH for 10 min followed by extensive washout. Thereafter, a nonselective PDE inhibitor (IBMX) was added to probe the PDE activity. (B) For comparison, the effect of IBMX was evaluated on thyroid follicles that were not previously stimulated with TSH. Shown are representative traces from four to six experiments per condition.

Mentions: In theory, the incomplete restoration of cAMP levels observed after prolonged TSH stimulation might have been due to an inactivation of PDEs. To investigate this possibility, we added a nonselective PDE inhibitor (IBMX) at the end of the washout phase. In contrast to control follicles that were not previously stimulated with TSH, on which IBMX had only a marginal effect, IBMX treatment caused a robust increase in cAMP levels when applied after TSH stimulation for 10 min and subsequent washout (Figure 6). These results demonstrated that, after extensive washout from a prolonged TSH stimulus, PDEs were still highly functional. Thus, it is unlikely that an inactivation of PDEs was the cause of the incomplete recovery of cAMP levels. On the contrary, the fast and robust increase of cAMP levels after IBMX addition suggested that the receptor-adenylyl cyclase system was indeed continuing to signal.

Calebiro D, Nikolaev VO, Gagliani MC, de Filippis T, Dees C, Tacchetti C, Persani L, Lohse MJ - PLoS Biol. (2009)

Bottom Line: TSH stimulation caused internalization of the TSH receptors into a pre-Golgi compartment in close association with G-protein alpha(s)-subunits and adenylyl cyclase III.Receptors internalized together with TSH and produced downstream cellular responses that were distinct from those triggered by cell surface receptors.These data suggest that classical paradigms of GPCR signaling may need revision, as they indicate that cAMP signaling by GPCRs may occur both at the cell surface and from intracellular sites, but with different consequences for the cell.

Affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany. davide.calebiro@yahoo.it

Abstract: G-protein-coupled receptors (GPCRs) are generally thought to signal to second messengers like cyclic AMP (cAMP) from the cell surface and to become internalized upon repeated or prolonged stimulation. Once internalized, they are supposed to stop signaling to second messengers but may trigger nonclassical signals such as mitogen-activated protein kinase (MAPK) activation. Here, we show that a GPCR continues to stimulate cAMP production in a sustained manner after internalization. We generated transgenic mice with ubiquitous expression of a fluorescent sensor for cAMP and studied cAMP responses to thyroid-stimulating hormone (TSH) in native, 3-D thyroid follicles isolated from these mice. TSH stimulation caused internalization of the TSH receptors into a pre-Golgi compartment in close association with G-protein alpha(s)-subunits and adenylyl cyclase III. Receptors internalized together with TSH and produced downstream cellular responses that were distinct from those triggered by cell surface receptors. These data suggest that classical paradigms of GPCR signaling may need revision, as they indicate that cAMP signaling by GPCRs may occur both at the cell surface and from intracellular sites, but with different consequences for the cell.