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).