Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors

Abstract

G protein–coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gαq and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.

A new kinetic method for measuring agonist efficacy and ligand bias using high resolution biosensors and a kinetic data analysis framework

“…One ligand was unbiased, relative to AngII (TRV055, Table 2). These results are consistent with the known bias profiles of these ligands 46,54,55 . For example, SII is an established arrestin-biased AT1 receptor ligand, and TRV055 is known as a balanced ligand (relative to AngII).…”

“…Arrestin recruitment to GPCRs is the first step in a signaling pathway that mediates myriad GPCR responses 26,27 that are often selectively activated over G-protein pathways by biased agonists 10,15,36,54,55 . Here we developed a new method of quantifying arrestin interaction that takes into account the kinetics, i.e.…”

“…Here kτ is used to assess signaling bias of the AT1 receptor ligands described above. These ligands have been reported to vary in their bias, with SII, TRV026 and TRV045 being arrestin biased 54,55 and TRV055 being unbiased 46 relative to AngII 46 . In this study, G-protein and arrestin signaling was measured using the same fluorescent biosensor assay modality, enabling near-identical conditions to be employed in comparing the pathways.…”

BRET sensors unravel thatPlasmodium falciparumserpentine receptor 12 (PfSR12) increases surface expression of mammalian GPCRs in HEK293 cells

“…The DAG biosensor is a unimolecular BRET-based sensor used to detect the formation of diacylglycerol in the plasma membrane. It is composed of a protein chimera containing a domain that attaches the biosensor to the membrane through myristoylation/palmitoylation, a GFP (acceptor), a long flexible linker, RLuc (donor) and a DAG interaction domain c1b from PKCδ (Figure 2A) (Namkung et al 2018).…”

“…The PKC biosensor is a construct of an RLuc conjugated to two PKC phosphorylation sites (TLKI and TLKD) linked by a flexible linker to phosphotreonine interaction domains (FHA1 and FHA2) and a GFP (Namkung et al 2018) ( Figure 4A).…”