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

The angiotensin type 2 (AT(2)) receptor and the bradykinin type 2 (B(2)) 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 AT(2) receptor and the B(2) 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 AT(2) receptors only occurred upon B(2) receptor coexpression and activation, all of which is indicative of AT(2)-B(2) receptor heteromerization. Additionally, we also observed specific coupling of the B(2) 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 B(2) receptor but not AT(2) receptor antagonism, indicating the importance of B(2) receptor activation within AT(2)-B(2) heteromers. The close proximity between the AT(2) receptor and B(2) receptor at the cell surface was also demonstrated with the NanoBRET ligand-binding assay. Together, our data demonstrate functional interaction between the AT(2) receptor and B(2) receptor in HEK293FT cells, resulting in novel pharmacology for both receptors with regard to Gα(q)/GRK2/β-arrestin2 recruitment (AT(2) receptor) and Gα(z) protein coupling (B(2) receptor). Our study has revealed a new mechanism for the enigmatic and poorly characterized AT(2) receptor to be functionally active within cells, further illustrating the role of heteromerization in the diversity of GPCR pharmacology and signaling.