Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors.
Sci Signal
; 11(559)2018 12 04.
Article
in En
| MEDLINE
| ID: mdl-30514808
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 ß-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 ß-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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Angiotensin II
/
Biosensing Techniques
/
GTP-Binding Proteins
/
Receptor, Angiotensin, Type 1
/
Bioluminescence Resonance Energy Transfer Techniques
/
Beta-Arrestins
Limits:
Animals
/
Humans
Language:
En
Journal:
Sci Signal
Journal subject:
CIENCIA
/
FISIOLOGIA
Year:
2018
Document type:
Article
Affiliation country:
Canada