Abstract Disclosure: E.K. Johnstone: Grant Recipient; Self; Australian Research Council. K.D. Pfleger: Grant Recipient; Self; Australian Research Council. Stock Owner; Self; Dimerix Bioscience Pty Ltd. Background: Receptor heteromerization is the phenomenon whereby two different receptors form a functional complex that attains unique pharmacological properties. Consequently, receptor heteromers can be considered novel drug targets, with the potential to achieve greater therapeutic selectivity and specificity. In a landmark study, the first receptor heteromer to be associated with a disease was the heteromer that forms between the angiotensin II type 1 (AT1) receptor and the bradykinin type 2 (B2) receptor (1). The AT1-B2 heteromer reportedly led to increased angiotensin II signalling, which was implicated in the angiotensin II hypersensitivity involved in pre-eclampsia. However, the validity of this study was questioned by a collaboration of several groups who were unable to find any evidence for a physical or a function interaction between the two receptors (2). As a consequence of these conflicting studies, the existence of the AT1-B2 receptor heteromer has remained controversial. Aim: Investigation of evidence for heteromerization of the AT1 receptor and the B2 receptor. Methods: AT1 receptor and B2 receptor pharmacology and potential heteromerization was investigated in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, as well as Dimerix’s Receptor-Heteromer Investigation Technology (Receptor-HIT) (3). Receptor-HIT enables the investigation of receptor heteromers through their specific ligand-induced proximity to interacting proteins. BRET assays, including in the Receptor-HIT configuration, allow real time, live cell monitoring of proximity between biomolecules of interest. Results: Using the Receptor-HIT assay, we confirmed the existence of the AT1-B2 receptor heteromer in HEK293FT cells. We found that the heteromer was able to recruit various GPCR interacting proteins, such as G proteins and arrestin in a bradykinin-dependent manner. Additionally, the heteromer also internalised and trafficked through the cell upon treatment with bradykinin. The close proximity of the two receptors was confirmed using a variation of the Receptor-HIT assay, which monitors receptor-ligand binding rather than protein-protein interactions. Discussion: Our study supports the existence of the AT1-B2 receptor heteromer and identifies various pharmacological properties of the complex. Further studies are required to relate these molecular properties to physiological consequences of heteromerization. (1)AbdAlla et al. Nat Med2001, 7 (9), 1003 1009.(2)Hansen et al. J Biol Chem2009;284(3):1831 1839.(3)Johnstone et al. Biochem Soc T. 2021;49:1555–65. Presentation: Thursday, June 15, 2023