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.