Tools to monitor the initial steps in GPCR signalling
Ligand binding to a GPCR induces a conformational change that enhances the affinity of the receptor for G proteins (Ghanouni et al., 2001). Upon G protein binding, GDP is exchanged for GTP which is present at a higher concentration in the cytoplasm (Bos et al., 2007), initiating G protein activation and further downstream signalling. Soon after RET-based cAMP sensors were created, the first RET biosensor detecting G protein signalling in living Dictyostelium discoideum cells was reported by Janetopoulos et al. (Janetopoulos et al., 2001). In this study, the activation of cAMP chemoattractant receptors was investigated by fusing Gα2-subunit to CFP and Gβ-subunit to YFP. Upon addition of chemoattractant cAMP, the Gα and Gβ subunits dissociate causing a reduction in FRET (Janetopoulos et al., 2001). This was an influential study that inspired other groups to interrogate the dynamics of GPCR–G protein coupling in eukaryotic cells. Initially, studies tested dissociation of the CFP- and YFP-tagged Gαβγ heterotrimer following agonist stimulation of the unmodified receptor (Azpiazu and Gautam, 2004). Further studies measured ligand-induced interaction between the GPCR of interest and Gα or Gβγ (Philip et al., 2007, Nobles et al., 2005, Hein et al., 2006). Analogous concepts were utilised for the development of BRET-based assays (Gales et al., 2005, Audet et al., 2008).
FRET/BRET changes between a GPCR and a G protein upon GPCR activation can be relatively small (Wan et al., 2018). Consequently, novel biosensors without membrane anchors were designed to generate higher RET changes upon GPCR activation. Among this type of sensor are conformation-specific nanobodies – binding domains derived from single chain camelid antibodies (Rasmussen et al., 2011). Originally, nanobodies were designed to assist GPCR crystallisation by stabilising the active conformation of a specific GPCR (Rasmussen et al., 2011). However, nanobodies can also be used as reporters of GPCR activation (Irannejad et al., 2013). When expressed in living cells, nanobodies translocate from the cytoplasm to their specific GPCR after it transitions into an active conformation (Irannejad et al., 2013). By fusion with fluorescent proteins, nanobodies can be used both in imaging and FRET/BRET-based assays to assess receptor activation (Figure 2A) (Irannejad et al., 2013) – an approach that has greatly advanced our understanding of endosomal GPCR signalling.
Within a pivotal study, Irannejad et al. used Nb80-GFP, a nanobody specific for the active conformation of the β2adrenoceptor (β2AR), to reveal that β2AR has a first wave of activation at the plasma membrane, and then a second wave of activation at endosomes (Irannejad et al., 2013). Using another nanobody (Nb37) against active Gαs, they further validated that G proteins are activated within this compartment (Figure 2B) (Irannejad et al., 2013). A similar approach based on Nb37 was used by Godbole et al. giving evidence that the TSH receptor activates endogenous Gαs proteins on membranes of the trans-Golgi network (Godbole et al., 2017). The resulting localised cAMP production and PKA activation close to the nucleus was required for efficient TSH-dependent CREB phosphorylation and gene transcription (Godbole et al., 2017).