A novel bioassay for detecting GPCR heterodimerization: transactivation of beta 2 adrenergic receptor by bradykinin...

Many G-protein-coupled receptors (GPCRs) have been shown to form heteromeric complexes primarily by biochemical methods, including competitive radioligand binding assays or measurements of changes in second-messenger concentration in lysed cells. These results are often cell line specific, and the expression of other cell surface proteins makes it difficult to detect potential functional consequences of GPCR interaction. Here, 2-electrode voltage clamping in Xenopus oocytes was used as a bioassay to explore heterodimerization of bradykinin type 2 receptor (Bk2R) and beta 2 adrenergic receptor (beta(2)AR), using chloride channels as outputs for receptor activation. The data show for the first time that these 2 receptors heterodimerize with functional consequences. Stimulation with bradykinin induced activation of Galphaq- and transactivation of Galphas-coupled pathways in oocytes expressing Bk2R and beta(2)AR. To corroborate these data, potential receptor interaction was examined in PC12 cells, a cell line that endogenously expresses both receptors, and confirmed that stimulation with bradykinin transactivates beta(2)AR. In both oocytes and PC12 cells, transactivation was ablated by Bk2R or beta(2)AR inverse agonists, suggesting that transactivation occurred directly through both receptors. This is the first evidence of Bk2R/beta(2)AR physical interaction, forming a functional heterodimer. The oocyte system may prove highly useful for exploration of GPCR heterodimerization and the functional consequences thereof.