A Novel Bioassay for Detecting GPCR Heterodimerization

Abstract

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 (β2AR), 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 Gαq- and transactivation of Gαs-coupled pathways in oocytes expressing Bk2R and β2AR. 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 β2AR. In both oocytes and PC12 cells, transactivation was ablated by Bk2R or β2AR inverse agonists, suggesting that transactivation occurred directly through both receptors. This is the first evidence of Bk2R/β2AR physical interaction, forming a functional heterodimer. The oocyte system may prove highly useful for exploration of GPCR heterodimerization and the functional consequences thereof.