The future of RET sensors
Investigating the physiological relevance of endosomal GPCR signalling
remains challenging. Dissecting local signals originating from the
plasma membrane versus local signals from intracellular compartments is
far from trivial and often requires the combination of multiple
FRET/BRET-based measurements and physiological readouts. An example of
where this has been demonstrated successfully is for the Neurokinin 1
receptor (NK1R). Jensen et al. reported that the
NK1R produces sustained signals from endosomes via
Gαq, generating signalling cascades that induce
nociception (Jensen et al., 2017). Within this study, the group used
FRET sensors tethered to the plasma membrane, nucleus, and cytosol to
show the significance of endosomal NK1R signalling on
extracellular signal-regulated kinase (ERK), cAMP, and protein kinase C
(PKC) signalling (Jensen et al., 2017). Substance P induced activation
of the aforementioned second messengers could be abolished by inhibiting
receptor internalisation (Jensen et al., 2017). Using a BRET-based
approach, the group subsequently found that activation of the receptor
with Substance P induced receptor trafficking away from the plasma
membrane into early endosomes, where the internalised receptor was able
to recruit Gαq, suggestive of G protein dependent
signalling by the NK1R at endosomes (Jensen et al.,
2017). To confirm the physiological relevance of this phenomenon, the
group employed endocytosis inhibitors to block NK1R
internalisation and observed that receptor internalisation was required
for sustained Substance P induced excitation of spinal neurons (Jensen
et al., 2017). In addition, a cholestanol conjugated
NK1R antagonist, designed to concentrate in endosomes,
was used to validate the specificity of this effect to endosomes (Jensen
et al., 2017). This study, and others (Yarwood et al., 2017,
Jimenez-Vargas et al., 2020), propose that pharmacological targeting of
certain endosomal GPCRs could offer improved and more selective
treatments for chronic pain.
Although internalisation inhibitors can be helpful for understanding the
effect of internalisation on GPCR signalling, the use of caged
agonists/antagonists or protein inhibitors of the GPCR signalling
machinery to block downstream responses may help to further investigate
intracellular signalling in a more detailed manner. Nanobodies have been
used to inhibit GPCRs at selective compartments like the Golgi
(Irannejad et al., 2017), and additional tools are being created to
block specific G protein isoform subtypes at subcellular compartments,
e.g. by tethering the regulator of G protein signalling (RGS) domain of
GRK2 to the plasma membrane or the early endosomes to block
Gαq downstream signalling (Wright et al., 2021). It is
likely that we will see the development of further toolsets to
selectively block GPCR signalling at specific subcellular compartments
and, thus, enable a robust interrogation of intracellular signalling and
their effects on physiological outputs.
In addition, improving our understanding of endogenous GPCR activity in
native cellular systems is essential. Many assays currently rely on the
overexpression of receptors in simple, easily transfectable, cell types.
However, signalling can be strongly influenced by cellular context.
There are some tools available that enable the detection of endogenous
GPCR activation. BRET sensors based on an ER/K linker and YFP (BERKY)
are an example of such emerging tools (Maziarz et al., 2020). Within
these sensors, the BRET donor and acceptor modules (NLuc and YFP,
respectively) are separated by a 10 nm-long ER/K α helix linker. On
opposite ends of the biosensor lie a membrane anchoring sequence
(N-terminus) and an active G protein detector module (C-terminus)
(Maziarz et al., 2020). Given the stochastic bending properties of the
ER/K α linker and the fact that G protein activation occurs on cell
membranes, these biosensors adopt a bent confirmation when the detector
module binds to active G proteins on membranes, increasing BRET (Maziarz
et al., 2020). This enables BERKY sensors to recognise subtype specific
Gα GTP as well as detect endogenous Gα protein activation (Maziarz et
al., 2020). BERKY sensors have been shown to detect subtype specific G
protein activation after stimulation of endogenous opioid and muscarinic
receptors in primary neurons (Maziarz et al., 2020). With further
modifications, sensors like this could be used to detect endogenous GPCR
activation at subcellular compartments.
Endogenous GPCR activity has also been detected by using
CRISPR/Cas9-mediated homology-directed repair to add NLuc (C-terminally)
onto the receptor of interest, thus facilitating the assessment of GPCR
activity under endogenous expression. White et al. used this approach to
detect CXC motif chemokine receptor 4 (CXCR4) internalisation,
trafficking, and β-arrestin recruitment in HEK293 cells (White et al.,
2017). Such methods could also be applicable to more physiologically
relevant cell/tissue types via knock-in pluripotent stem cells or even
knock-in animals (Merkle et al., 2015).
In summary, RET sensors have dramatically changed our understanding of
how GPCRs signal spatially and temporally. They have allowed real-time
monitoring of receptor coupling, trafficking, and second messenger
activity in live cells, which have all been instrumental towards our
understanding of endosomal signalling. If, in the future, we could
utilise these methods and other complementary approaches, to investigate
endogenous receptors in their native cell types or even in vivo ,
we should gain a much clearer understanding of the physiological
relevance of GPCR signalling at intracellular sites. Not only will such
research help to untangle a key emerging mechanism of GPCR signalling,
but it may permit more targeted therapeutic approaches for a variety of
diseases.