Monitoring agonist-promoted conformational changes of beta-arrestin in living cells by intramolecular BRET

Abstract

Recruitment of beta-arrestin (beta-arr) to agonist-stimulated G-protein-coupled receptors (GPCRs) has a crucial role in controlling signalling efficacy and selectivity. When translocated to the receptor, beta-arr is believed to undergo important conformational rearrangement necessary for its downstream actions. To probe these changes in living cells, we constructed an intramolecular bioluminescence resonance energy transfer (BRET)-based biosensor, in which beta-arr is sandwiched between the Renilla luciferase (Luc) and the yellow fluorescent protein (YFP). We show that the intramolecular BRET between Luc and YFP was significantly increased following GPCR activation, suggesting a conformational rearrangement bringing the amino terminus and carboxyl terminus of beta-arr in closer proximity. Kinetic analysis showed that this conformational change follows the initial beta-arr/receptor engagement. In addition to providing new insights into the agonist-induced conformational rearrangements of beta-arr in living cells, the double-brilliance beta-arr offers a universal biosensor for GPCR activation, allowing the study of native receptors in large-scale screening analysis.

Figure 1

Double-brilliance β-arr. Schematic diagram illustrating how agonist-promoted conformational rearrangement of β-arr can be measured as changes in BRET using double-brilliance β-arr. Luc and YFP are represented by cylinders proportional to their sizes, but their real orientation is unknown.

Figure 2

Functionality of double-brilliance β-arr. HEK293 (A–C) or COS (D) cells were transiently transfected with the indicated plasmids. (A) Cells incubated or not in the presence of saturating concentrations of specific agonists (β₂-AR, 10 μM isoproterenol (ISO); V2R, 1 μM arginine vasopressin (AVP)). Localization of Luc–β-arr–YFP and Myc-tagged receptors was analysed by confocal fluorescence microscopy. (B) Agonist-induced recruitment of β-arr measured using BRET². t_1/2=half-time of maximal β-arr recruitment. (C) Dose-dependent recruitment of β-arr to the receptors measured in BRET² following 2 min stimulation with the agonist. EC₅₀=concentration of agonist producing half-maximal β-arr recruitment. (D) Cells treated or not for 15 min with the specific agonists at 37°C and cellsurface receptor levels measured by enzyme-linked immunosorbent assay (ELISA). Receptor endocytosis is defined as the loss of cell-surface immunoreactivity and is expressed as a percentage of total immunoreactivity measured under basal conditions. Expression levels of β-arr were controlled using western blot (data not shown). Data are the mean±s.e.m. of at least three independent experiments. ^*P<0.05 between treatment and each individual control condition. Mock, nontransfected cells.

Figure 3

AVP-induced conformational change of β-arr monitored by intramolecular BRET. HEK293 cells were transfected with the indicated plasmids and BRET was measured at 25°C in the presence of coelenterazine h. (A) Specificity of agonist-induced β-arr intramolecular BRET. (B) Real-time BRET measurements of the agonist-induced β-arr conformational change. t_1/2=half-time of maximal conformational change of β-arr. (C) Dose-dependent agonist-promoted increase of β-arr intramolecular BRET. Cells were stimulated with increasing concentrations of AVP for 4 min. EC₅₀=concentration of AVP producing half-maximal conformational change of β-arr. Data are the mean±s.e.m. of at least three independent experiments. ^*P<0.01 between treated and control condition.

Figure 4

Agonist-promoted conformational change of β-arr is independent of receptor phosphorylation. HEK293 cells were transfected with V2R and either Luc–β-arr–YFP or Luc–β-arr(R169E)–YFP. Cells were stimulated or not for 10 min in the presence of 1 μM AVP. Inset: AVP-induced BRET increase. Data are the mean±s.e.m. of three independent experiments. ^*P<0.02 between treatment and each individual control condition.

Figure 5

Double-brilliance β-arr monitors GPCR activation. HEK293 cells were transfected with Luc–β-arr–YFP and either pcDNA3.1 or plasmids encoding the indicated receptors. (A) Agonist-induced translocation of Luc–β-arr–YFP measured following treatment with 1 μM of the specific agonists (β₂-AR, ISO; V1aR, AVP; δ-OR, SNC80; PAFR, PAF; CCR5, hRANTES; AT1aR, angiotensin II). (B) Agonist-induced conformational change of Luc–β-arr–YFP measured following 10 min stimulation with the specific agonists mentioned in (A). Data are the mean±s.e.m. of three independent experiments. ^*P<0.05 between treatment and each individual control condition.