GPCR trafficking
RET methods can also be used to investigate GPCR trafficking and further
validate the presence of GPCRs at subcellular compartments with high
spatiotemporal resolution (Figure 2C). Zacharias et al. introduced the
use of FRET to monitor clustering of acylated proteins in the plasma
membrane (Zacharias et al., 2002). Using a similar approach, Drake et
al. subsequently followed the internalisation of the
β2AR by measuring the FRET decrease between CFP tagged
β2AR and plasma membrane-tethered mYFP (Drake et al.,
2008). Subsequent groups have applied this principle to BRET-based
techniques, measuring agonist-induced BRET between a luciferase-tagged
GPCR and fluorescently tagged intracellular compartment markers. As BRET
offers an improved signal-to-noise ratio compared to FRET, BRET
approaches have been used more extensively to characterise GPCR
trafficking to various membrane compartments.
In 2011, Lan et al. showed translocation of the β2AR
from the plasma membrane to early endosomes by monitoring ligand-induced
BRET changes between β2AR-RLuc8 and Venus-tagged K-Ras
(plasma membrane marker) or Venus-tagged Rab5 (early endosome marker)
(Lan et al., 2011). In this study, the group were able to demonstrate
that only active β2ARs internalise, and that
associations between β2AR protomers are likely transient
(Lan et al., 2011). Soon after, the same group used this methodology for
the investigation of protein localisation at other subcellular
locations. They demonstrated that β2AR traffics through
various endosomal compartments, as well as the ER and Golgi (Lan et al.,
2012). In addition, they showed the applicability of BRET to determine
outer versus inner membrane protein topology at the plasma membrane, ER,
and mitochondria (Lan et al., 2012).
In 2016, this methodology was used to validate the significance of
clinically relevant Vasopressin 2 receptor (V2R)
mutations (Tiulpakov et al., 2016). Further Venus-tagged intracellular
compartment markers were validated and added to the previously described
toolset to facilitate a yet more detailed understanding of GPCR
trafficking via BRET. This included markers of ER to Golgi trafficking
(Rab1, Rab6), trans-Golgi trafficking (Rab8), and fast/slow endosomal
recycling (Rab4/Rab11) (Tiulpakov et al., 2016). Using this BRET-based
technique, the group were able to validate subtle differences in the
trafficking profiles of distinct V2R mutants, giving
further insight into the mechanisms behind nephrogenic syndrome of
inappropriate antidiuresis (NSIAD) and nephrogenic diabetes insipidus
(Tiulpakov et al., 2016).
BRET has also been shown to be capable of high-throughput endosomal GPCR
trafficking assays using plate reader-based detection (Giubilaro et al.,
2021). Using such an approach, Giubilaro et al. investigated the
properties of biased compounds and their effects on GPCR localisation
and trafficking. This led to the identification of a novel Ras and ARF6
inhibitor (Rasarfin), capable of blocking the internalisation of the
angiotensin II type 1 receptor (AT1R) and other GPCRs. Rasarfin may have
applications as an anti-proliferative agent e.g. for use as an inhibitor
of oncogenic cellular responses (Giubilaro et al., 2021).