Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context

Abstract

Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gα, Gα, Gα, and Gα or β-arrestins, we showed that Gα and Gα are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin.

Distinct Conformational Dynamics of Three G Protein-Coupled Receptors Measured Using FlAsH-BRET Biosensors

“…Responses in ICL3 in AT1R and FP were both robust and sustained (Figures 6D,E) compared to the β 2 AR. Further, robust sustained responses have also been detected in both ICL2 and the C-terminus of AT1R [(17); Figure 6F]. Interestingly, no responses were detected in similar constructs built into either ICL2 or the C-tail of FP (data not shown).…”

“…However, the responses in the other C-tail sensors were much more robust and sustained (Figures 6B,C). We have previously analyzed responses to ligand in both FP (11) and in the AT1R (17). Responses in ICL3 in AT1R and FP were both robust and sustained (Figures 6D,E) compared to the β 2 AR.…”

“…This pattern was distinct in the AT1R and in FP receptor. The AT1R exhibits the most conformational heterogeneity in that sensors engineered into ICL2, ICL3, or the C-tail all reported robustly on conformational changes in response to either canonical (Ang II or Ang III) or biased (SI) ligands (17). Further, only ICL3 biosensors reported responses upon stimulation with PGF2α in FP (11, data not shown for ICL2 or the C-tail).…”

“…The recombinant receptors used in this paper are as follows: SP-FLAG-hAT1R-CCPGCC-ICL3-p3-RlucII or SP-FLAG-hAT1-R-CCPGCC-C-tail-p1-RlucII in a pIRESH plasmid backbone (17) along with SP-HA-hFP-CCPGCC-ICL3-p4-RlucII in a pcDNA3.1(−) backbone (11), in addition to the panel of eight β 2 AR biosensors expressed in a pIRESpuro3 plasmid backbone.…”

“…We have explored the use of FlAsH BRET in the conformational profiling of the prostaglandin F2α receptor [FP; (11)], and the angiotensin II type I receptor [AT1R; (17)]. Here, we introduced this tetracysteine tag at various locations within the coding sequence of the β 2 AR in order to report on conformational changes upon agonist stimulation.…”