Conformational profiling of the AT 1 angiotensin II receptor 1 Conformational profiling of the AT 1 angiotensin II receptor reflects biased agonism , G protein coupling and cellular context

Here, we report the design and use of GPCR-based biosensors to monitor ligandmediated conformational changes in receptors in intact cells. These biosensors use Bioluminescence Resonance Energy Transfer (BRET) with Renilla 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 carboxyterminal tail of the angiotensin II type I receptor (AT1R). 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 CRISPR/Cas9-engineered to delete Gαq, Gα11, Gα12, and Gα13 or β-arrestins, we showed that Gαq and Gα11 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 contextdependent readouts of GPCR conformation. Our study provides mechanistic insights into signalling events that depend on either G proteins or β-arrestin. G protein-coupled receptors (GPCRs) constitute the largest class of membrane receptors. Encoded by more than 800 genes in the human genome, they represent targets of a variety of clinically used drugs. A single GPCR occupied by its cognate orthosteric or by allosteric ligands can trigger a complex array of signal transduction pathways which can in some cases be selectively modulated through development and use of biased ligands (1). Such molecules can modulate a subset of the total signalosome, likely by inducing distinct http://www.jbc.org/cgi/doi/10.1074/jbc.M116.763854 The latest version is at JBC Papers in Press. Published on February 17, 2017 as Manuscript M116.763854 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Feruary 9, 2017 hp://w w w .jb.org/ D ow nladed from by gest on Feruary 9, 2017 hp://w w w .jb.org/ D ow nladed from by gest on Feruary 9, 2017 hp://w w w .jb.org/ D ow nladed from by gest on Feruary 9, 2017 hp://w w w .jb.org/ D ow nladed from by gest on Feruary 9, 2017 hp://w w w .jb.org/ D ow nladed from by gest on Feruary 9, 2017 hp://w w w .jb.org/ D ow nladed from by gest on Feruary 9, 2017 hp://w w w .jb.org/ D ow nladed from Conformational profiling of the AT1 angiotensin II receptor 2 conformational changes in GPCR structure which translates into differential effector engagement. The development of such biased molecules, aside from being powerful tools to study GPCR signalling, might also lead to clinically relevant compounds with better efficacy and side-effect profiles. To understand how pervasive functional selectivity is, and how it might be exploited for therapeutic purposes, an increasing number of studies focus on obtaining signalling signatures by measuring larger and larger numbers of signalling pathways potentially modulated by panels of receptor ligands (2-9). Such approaches are very useful as they can be used to identify novel pathways downstream of GPCRs, capture the protean nature of receptor agonism (i.e. that in some cases antagonists act like agonists and vice versa) and can often link both therapeutic and adverse consequences to particular signalling pathways. However, when the relevant signalling pathways in a given cell type are incompletely understood, such profiles may be incomplete. Also, it is possible that the signalosome downstream of particular receptors may be different in distinct cells types raising the issue of portability of signalling sensor platforms (10). Structurally, GPCRs are characterized by an extracellular N-terminal tail, followed by seven transmembrane α-helices connected by three intracellular (ICL1-3) and three extracellular loops (ECL1-3), ending with an intracellular C-terminal tail (C-tail). GPCRs fold themselves into a barrel-like structure, with the seven transmembrane helices forming a cavity that serves in many cases as a ligand-binding domain. There are many optical approaches being used to understand GPCR signalling, interactions and conformational dynamics (reviewed in (11,12)). Previous studies have shown that engineering FlAsH-binding sequences into different positions in GPCRs with FRET or BRET partners such as YFP or Renilla luciferase can be used to produce biosensors that report on ligand-induced conformational changes in receptors (13-18) or downstream effectors (19-21). In this regard, we have engineered several GPCR-based biosensors to monitor ligand-mediated conformational changes in intact HEK 293 cells and in vascular smooth muscle cells from distinct vantage points. A set of biosensors was generated for the angiotensin II (Ang II) AT1 receptor (AT1R), a prototypical Gαq-coupled GPCR, where we examined responses to balanced and biased ligands (22) as well as the role of cell context in determining conformational outcomes. Combining such biosensor approaches with selective knockout of G proteins or β-arrestin isoforms using CRISPR/Cas9 offers insights into the role of receptor/G protein or receptor/βarrestin interactions in driving receptor conformational responses to ligands.