Characterisation of the Trafficking Profiles of Angiotensin II Receptor Heteromers Using Bioluminescence Resonance Energy Transfer

EKM Johnstone1, KDG Pfleger1,2. 1Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia, 2Dimerix Bioscience, Perth, Western Australia, Australia

Angiotensin II (AngII) is a vasoconstrictive hormone that acts upon both the AngII type 1 receptor (AT1R) and the AngII type 2 receptor (AT2R). Most of the well characterised biological actions of AngII are mediated by the AT1R, while the AT2R remains poorly characterised, though often believed to counteract many AT1R-mediated effects. Like many GPCRs, the concept of receptor heteromerisation has revealed new complexity in angiotensin II receptor signalling systems through the formation of receptor complexes with unique signalling and regulatory properties.

The aim of this study was to investigate the internalisation and trafficking profiles of the AT1R and the AT2R when coexpressed with the bradykinin type 2 receptor (B2R). Interactions between the AT1R and the B2R have been described1, but remain controversial2. Interactions between the AT2R and the B2R have also been demonstrated3, however the internalisation and trafficking properties of the complex have not been described. In this study, receptor trafficking profiles were monitored using an unconventional approach employing bioluminescence resonance energy transfer (BRET). Here, receptor proteins were tagged with the Rluc8 enzyme, while membrane associated protein markers were tagged with the Venus fluorophore4. The membrane proteins used were Venus-Kras (plasma membrane), Venus-Rab5 (early endosome), Venus-Rab7 (late endosome) and Venus-Rab11 (slow recycling endosome). Changes in the BRET signal upon ligand treatment enabled monitoring of cellular localization of the receptors. Addition of a second, untagged receptor into the system allowed comparisons between monomer/homomer and heteromer trafficking profiles. Statistical significance of the differences between conditions was calculated using two-way analysis of variance with Bonferroni post-test.

AngII treatment caused rapid internalisation and endosomal trafficking of the AT1R, and co-expression with the B2R did not significantly alter this trafficking profile (p > 0.05). Bradykinin (BK) treatment did not cause internalisation or endosomal trafficking of the AT1R, and although coexpression with the B2R appeared to result in a weak level of endosomal trafficking, this was not significant. Dual AngII and BK treatment caused the same level of internalisation and endosomal trafficking of the AT1R as AngII treatment alone (p > 0.05), however coexpression with the B2R caused a significantly reduced level of internalised receptors (p < 0.001) and less receptors observed in recycling endosomes (p < 0.01).

As expected for the AT2R, AngII treatment did not cause internalisation or endosomal trafficking, and coexpression with the B2R did not alter this profile. Likewise, BK treatment did not cause internalisation or endosomal trafficking of the AT2R, however coexpression with the B2R resulted in a highly significant increase in surface expression of the AT2R (p < 0.001). Additionally, BK treatment and B2R coexpression caused a significant increase in trafficking into late endosomes (p < 0.01).

In summary, coexpression and potential heteromerisation with the B2R causes alterations in the internalisation and trafficking profiles of both the AT1R and the AT2R. This study has also demonstrated the use of an unconventional approach for investigating receptor heteromer internalisation and trafficking.

[1] AbdAlla S et al, Nat Med 7:1003, 2001.

[2] Hansen JL et al, J Biol Chem 284:1831, 2009.

[3] Abadir P et al, Hypertension 48:316, 2006.

[4] Jensen DD et al, J Biol Chem 288:22942, 2013