Signal profiling of the βAR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by βAR and βAR.

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β-adrenergic receptor (βAR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR. These include coupling to G and G pathways. The signalling cascade linking the βAR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both G and G are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.