cAMP tools investigating spatiotemporal GPCR signalling
Genetically encoded cAMP/PKA sensors have greatly advanced our spatial and temporal understanding of cAMP signalling compared to endpoint biochemical methods. Their use has proven essential for the demonstration of compartmentalised GPCR signalling and, more recently, the discovery of GPCR signalling at intracellular sites.
In 2009, FRET-based Epac cAMP sensors were used by two independent groups to show that certain GPCRs can internalise and generate sustained cAMP signals at intracellular sites (Calebiro et al., 2009, Ferrandon et al., 2009). Using a transgenic mouse with ubiquitous expression of a FRET-based Epac cAMP biosensor, Calebiro et al. observed that the thyroid stimulating hormone (TSH) receptor can trigger persistent cAMP signals after internalisation (Calebiro et al., 2009). This phenomenon was further validated using subcellular fractionation methodologies and inhibitors of receptor endocytosis, which prevented persistent cAMP signalling (Calebiro et al., 2009). Intriguingly, it was later shown that sustained TSH receptor signalling is cell-type specific, occurring in primary thyroid cells but not in HEK293 cells (Werthmann et al., 2012).
In parallel, Ferrandon et al. demonstrated, using the same Epac cAMP FRET biosensor, that internalised parathyroid hormone (PTH) receptors are capable of persistent cAMP signals (Ferrandon et al., 2009). This sustained response was dependent on the agonist used as it was triggered by PTH1-34, but not by human parathyroid related peptide­ (PTHrP1-36), suggesting that the sustained response required a tighter interaction of the agonist with the receptor (Ferrandon et al., 2009).
Measurement of local cAMP and PKA production/activation can be achieved by tethering genetically encoded sensors to specific subcellular compartments. This approach has been applied to measure local cAMP and PKA responses induced by the TSH receptor in thyroid cells (Godbole et al., 2017). After internalisation, TSH receptors were found to be inactive in the early endosome compartment. However, after retrograde trafficking to the trans-Golgi network, the receptor was shown to mediate local cAMP/PKA signalling close to the nucleus (Godbole et al., 2017). By tethering Epac1-cAMP and AKAR2 sensors to the trans-Golgi network or the plasma membrane, it was shown that the TSH receptor has an internalisation-dependent, late-stage, intracellular signalling component that regulates downstream CREB phosphorylation and gene transcription via Gαs (Godbole et al., 2017).
Similar approaches have been used to target BRET-based cAMP sensors to subcellular compartments. For example, CAMYEL, a Epac-based BRET sensor that is normally present in the cytoplasm, has been compared to a plasma membrane-tethered CAMYEL to study the role of cytosolic and membrane-bound phosphodiesterase (PDE) subtypes, and their importance in the regulation of local cAMP concentrations (Matthiesen and Nielsen, 2011). Tethering CAMYEL, or other similar BRET-based sensors, to local compartments may help to further enhance our understanding of GPCR signalling at intracellular sites.
In addition, a complementary optogenetic approach has been employed to investigate the consequences of local cAMP/PKA signalling. This approach is based on a photoactivated adenylyl cyclase, bPAC, which produces cAMP upon stimulation with blue light (Tsvetanova and von Zastrow, 2014). By targeting bPAC to the cytoplasm, plasma membrane, or early endosomes, Tsvetanova et al. showed that cAMP production in the cytoplasm and early endosomes induces a greater transcriptional response than at the plasma membrane, giving further evidence that internalisation of at least some GPCRs is required to elicit full transcriptional responses. This study further suggests that endosomes can act as a shuttle system from the plasma membrane to intracellular sites, to enhance the efficiency of GPCR signalling (Tsvetanova and von Zastrow, 2014).