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).