Novel Pharmacology Following Heteromerization of the Angiotensin II Type 2 Receptor and the Bradykinin Type 2 Receptor

Elizabeth K M Johnstone; Mohammed Akli Ayoub; Rebecca J Hertzman; Heng B See; Rekhati S Abhayawardana; Ruth M Seeber; Kevin D G Pfleger

doi:10.3389/fendo.2022.848816

Novel Pharmacology Following Heteromerization of the Angiotensin II Type 2 Receptor and the Bradykinin Type 2 Receptor

Front Endocrinol (Lausanne). 2022 May 26:13:848816. doi: 10.3389/fendo.2022.848816. eCollection 2022.

Authors

Elizabeth K M Johnstone^{1

2

3}, Mohammed Akli Ayoub^{1

4}, Rebecca J Hertzman^{1

3}, Heng B See^{1

2}, Rekhati S Abhayawardana^{1

2}, Ruth M Seeber^{1

2}, Kevin D G Pfleger^{1

2

5}

Affiliations

¹ Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, Nedlands, WA, Australia.
² Australian Research Council Centre for Personalised Therapeutics Technologies, Perth, WA, Australia.
³ School of Biomedical Sciences, The University of Western Australia, Nedlands, WA, Australia.
⁴ Department of Biology, College of Science, United Arab Emirates University, Al Ain, United Arab Emirates.
⁵ Dimerix Limited, Nedlands, WA, Australia.

Abstract

The angiotensin type 2 (AT₂) receptor and the bradykinin type 2 (B₂) receptor are G protein-coupled receptors (GPCRs) that have major roles in the cardiovascular system. The two receptors are known to functionally interact at various levels, and there is some evidence that the observed crosstalk may occur as a result of heteromerization. We investigated evidence for heteromerization of the AT₂ receptor and the B₂ receptor in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, including the Receptor Heteromer Investigation Technology (Receptor-HIT) and the NanoBRET ligand-binding assay. The Receptor-HIT assay showed that Gα_q, GRK2 and β-arrestin2 recruitment proximal to AT₂ receptors only occurred upon B₂ receptor coexpression and activation, all of which is indicative of AT₂-B₂ receptor heteromerization. Additionally, we also observed specific coupling of the B₂ receptor with the Gα_z protein, and this was found only in cells coexpressing both receptors and stimulated with bradykinin. The recruitment of Gα_z, Gα_q, GRK2 and β-arrestin2 was inhibited by B₂ receptor but not AT₂ receptor antagonism, indicating the importance of B₂ receptor activation within AT₂-B₂ heteromers. The close proximity between the AT₂ receptor and B₂ receptor at the cell surface was also demonstrated with the NanoBRET ligand-binding assay. Together, our data demonstrate functional interaction between the AT₂ receptor and B₂ receptor in HEK293FT cells, resulting in novel pharmacology for both receptors with regard to Gα_q/GRK2/β-arrestin2 recruitment (AT₂ receptor) and Gα_z protein coupling (B₂ receptor). Our study has revealed a new mechanism for the enigmatic and poorly characterized AT₂ receptor to be functionally active within cells, further illustrating the role of heteromerization in the diversity of GPCR pharmacology and signaling.

Keywords: BRET; GPCR; NanoBRET; angiotensin receptor; bradykinin receptor; heteromer; receptor-HIT.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bradykinin / pharmacology
Ligands
Receptor, Angiotensin, Type 2* / physiology
Receptor, Bradykinin B2* / physiology
Receptors, G-Protein-Coupled
beta-Arrestin 2

Substances

Ligands
Receptor, Angiotensin, Type 2
Receptor, Bradykinin B2
Receptors, G-Protein-Coupled
beta-Arrestin 2
Bradykinin