Nanobodies have also been used in combination with NanoBiT approaches to assess endosomal GPCR activation. McGlone et al. fused Nb37 with SmBiT (Nb37-SmBiT) and measured ligand-induced luminescence upon complementation with plasma membrane (CAAX-LgBiT) or early endosome (Endofin-LgBiT) tethered LgBiT, to investigate compartmentalised signalling of the glucagon receptor (GCGR) (McGlone et al., 2021). From this investigation, the group observed that the GCGR signals both at the plasma membrane and the endosomes. In addition, using imaging and luminescence methods, it was found that RAMP2 expression significantly increased intracellular GCGR presence and activity, but had little effect on plasma membrane GCGR activity. Using such methods, RAMP2 was shown to alter the spatiotemporal activity of the GCGR (McGlone et al., 2021).
Considering that GPCRs are now thought to signal from multiple subcellular membranes (Calebiro and Koszegi, 2019), one insufficiently understood aspect is how G proteins access such membranes and whether they are constitutively present at these compartments. BRET-based approaches have been used to investigate G protein activation and trafficking to subcellular compartments. In an elegant study, Martin et al. monitored the localisation and movement of G proteins in live cells (Martin and Lambert, 2016). By measuring BRET between Gαs-RLuc8 and Venus-tagged intracellular compartment, upon activation of co-expressed β2AR with isoproterenol, Gαs was shown to leave the plasma membrane and rapidly associate with the ER, mitochondria, and endosomes (Martin and Lambert, 2016). From these results, it was suggested that activation of Gαs causes the protein to lose some of its affinity for the plasma membrane, enter the cytosol, and sample intracellular membrane compartments (Martin and Lambert, 2016).
More recently, mini-G proteins, engineered Ras domains of Gα subunits, have been created to enhance our understanding of GPCR activation in live cells (Nehme et al., 2017, Wan et al., 2018). Mini-G proteins were designed to mimic all main G protein isoforms (Gαi/o, Gαs, Gαq, and Gα12), and are frequently used to investigate GPCR coupling specificity, ligand pharmacology, and spatiotemporal GPCR signalling (Figure 2) (Wan et al., 2018). Like nanobodies, mini-G proteins were originally used to improve the stability of GPCR–G protein complexes, enabling GPCR crystallisation for structural studies (Nehme et al., 2017). However, mini-G proteins can also be fused to fluorescent or luminescent proteins, as well as self-labelling protein tags, for imaging, FRET, BRET, and luminescence measurements (Wan et al., 2018). Since mini-G proteins are relieved from membrane attachments, they rapidly translocate from the cytoplasm to the active receptor upon stimulation, which can be visualised and measured via microscopy or RET-based approaches (Wan et al., 2018). For example, by monitoring agonist induced mini-Gαq translocation to fluorescently tagged early endosome markers, employing both imaging and BRET-based methodologies, Wright et al. showed that several GPCRs are activated at early endosomes (Wright et al., 2021). In addition, the study suggests that Gαq activation at endosomes requires a first activation event at the plasma membrane and that subsequent G protein activity at endosomes is either self-sustained or can be enhanced by endocytosed GPCR (Wright et al., 2021). Mini-G proteins have been used in a similar manner to measure endosomal activation of the glucagon-like peptide-1 receptor (GLP-1R) (Lucey et al., 2021) and Golgi activation of A1-adenosine receptors (Wan et al., 2018). The application of mini-G proteins has given novel insight into spatiotemporal GPCR signalling and will undoubtedly help to further characterise our understanding of intracellular GPCR signalling over the coming years.