Research ResourceGPCR SIGNALING

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

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Science Signaling  04 Dec 2018:
Vol. 11, Issue 559, eaat1631
DOI: 10.1126/scisignal.aat1631

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Biosensors for GPCR-biased signaling

Upon binding to ligands, G protein–coupled receptors (GPCRs) stimulate heterotrimeric G proteins and recruit β-arrestin, which dampens signaling through G proteins and acts as a scaffold to activate other pathways. Whereas some ligands stimulate both G proteins and β-arrestin, others preferentially stimulate one or the other, a phenomenon known as biased agonism. For receptors engaging more than one G protein subtype, ligands may also bias downstream signaling to a particular G protein. Namkung et al. developed bioluminescence resonance energy transfer (BRET) biosensors, which they used to characterize biased signaling downstream of the angiotensin II (AngII) type 1 receptor (AT1R) in response to various ligands, as well as the bias of naturally occurring AT1R variants. This tool kit can be used to comprehensively dissect the signaling bias of other GPCR-ligand combinations, which may benefit drug development efforts.


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.

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