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.