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

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 Gαq, 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 Gαz protein, and this was found only in cells coexpressing both receptors and stimulated with bradykinin. The recruitment of Gαz, Gαq, 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 Gαq/GRK2/β-arrestin2 recruitment (AT2 receptor) and Gαz 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.

Unraveling the crosstalk between renin‐angiotensin system receptors

“…86 Conversely, B 2 R couples to Gα z only in cells coexpressing both receptors and stimulated with bradykinin. 86 Gα z belongs to the Gα i/o class of G proteins, and its typical effect is the inhibition of adenylyl cyclase and cAMP signaling. Therefore, the heteromerization between AT 2 R and B 2 R may introduce novel G-protein coupling for both receptors.…”

“…2 However, in the presence of B 2 R, there is recruitment of Gα q , G-protein kinase 2, and β-arrestin2 to AT 2 R, indicating AT 2 R-B 2 R heteromerization. 86 Conversely, B 2 R couples to Gα z only in cells coexpressing both receptors and stimulated with bradykinin. 86 Gα z belongs to the Gα i/o class of G proteins, and its typical effect is the inhibition of adenylyl cyclase and cAMP signaling.…”

“…The AT 2 R is atypical among GPCRs as G‐protein coupling of the AT 2 R is unconventional, as it does not couple to G q , and G i coupling does not lead to cAMP inhibition 2 . However, in the presence of B 2 R, there is recruitment of Gα q , G‐protein kinase 2, and β‐arrestin2 to AT 2 R, indicating AT 2 R–B 2 R heteromerization 86 . Conversely, B 2 R couples to Gα z only in cells coexpressing both receptors and stimulated with bradykinin 86 .…”

GPCR heteromers: An overview of their classification, function and physiological relevance

“…Recently, we have discovered novel G protein coupling by the heteromer that forms between the angiotensin II AT 2 receptor and the bradykinin B 2 receptor. Although neither receptor recruits Gα z following treatment with agonist, upon their coexpression Gα z is recruited to the heteromer in a bradykinin dependent manner ( 57 ).…”