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).