β-arrestin1 and 2 exhibit distinct phosphorylation-dependent conformations when coupling to the same GPCR in living cells

Figures

• 

  Fig. 1. The configuration of specific PTH1R–β-arrestin complexes determines their functionality. a Schematic depiction of the ‘hanging’, ‘core’ and GRK-independent GPCR-β-arrestin complex configurations. b NanoBRET-measured recruitment of β-arrestin1 and 2 to the PTH1R upon stimulation with indicated concentrations of PTH(1-34). Curves show the recruitment measured for β-arrestin WT constructs in HEK293 cells (HEK-WT), analogous experiments performed in ΔQ-GRK cells and recruitment measurements of β-arrestin-dFLR mutants in HEK-WT. Results are shown as Δ net BRET fold change, mean of at least three independent repetitions (βarr-dFLR and βarr in ΔQ-GRK n = 3; βarr-WT n = 4) ± SEM. c Data correspond to the BRET changes at saturating ligand concentration from b, normalised to the respective β-arrestin WT condition. To test for significance between β-arrestin association in a ‘hanging’ or GRK-independent complex, a one-way ANOVA, followed by a two-sided Tukey’s test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). For both β-arrestin 1 and 2, WT vs respective βarr-dFLR and βarr in ΔQ-GRK p < 0.0001. d and e Representative live-cell confocal microscopy images of β-arrestin1/2…

• 

  Fig. 2. Generation and functionality of β-arrestin1 and 2 NanoBRET conformational change biosensors. a Overall sensor design of intramolecular β-arrestin1 and 2 conformational change biosensors. The NanoLuc BRET donor is genetically fused to the arrestin C-terminus and individual FlAsH-binding motifs are introduced at ten different positions in outward-facing loops of the arrestin N- and C-domains (F1 – F10, coloured loops and amino acid sequence positions denoted for β-arrestin-1 and 2). In the inactive state (PDB: 1CF1, with a modelled C-terminus depicted in ochre), the NanoLuc is in close proximity of the individual FlAsH-binding motifs, resulting in an efficient energy transfer. Upon GPCR- and subsequent arrestin activation (PDB: 5W0P), the distance between the BRET pair changes, depending on the nature of the conformational change and the specific labelling position. b Characterisation of β-arrestin1/2-F5 conformational change biosensors. Shown are the emission spectra of the F5 conformational change biosensors for β-arrestin1 and 2. The spectra were measured with and without FlAsH labelling (mock, blue), in the basal state (vehicle, green) and after ligand addition (0.3 µM…

• 

  Fig. 3. β-arrestin1 and 2 display different conformational change signatures upon coupling to the PTH1R. a Depiction of conformational changes induced by PTH1R activation for β-arrestin1 and 2 at 3 µM PTH(1-34) for sensors located in the respective N-domains. The β-arrestin conformational change data were generated by transfection of HEK-WT cells with an untagged PTH1R-WT expression construct and one β-arrestin conformational change biosensor, FlAsH-labelling and stimulation with 3 µM PTH(1-34). Results are shown as Δ net BRET change in percent, mean of at least three independent repetitions (β-arrestin2-F3 n = 4, all other sensors n = 3) ± SEM. b Radar chart representation of the data from a and c, normalised to the respective F10 biosensor values in each dataset, as this particular sensor shows similar conformational change signals for both β-arrestin isoforms. c Shows conformational change measurements of β-arrestin1 and 2 sensors located in the respective C-domains, analogous to the N-domain data presented in a. Results are shown as Δ net BRET change in percent, mean of at least three independent repetitions (β-arrestin1-F9 and β-arrestin2-F10 n = 4, all other sensors n = 3) ± SEM. d…

• 

  Fig. 4. The PTH1R phosphorylation state differentially affects recruitment of β-arrestin1 and 2 and the resulting complex configurations. a and b Overall recruitment and schematic depiction of our approach to assess phosphorylation-specific PTH1R β-arrestin activation. For the PTH1R-PD1 mutant receptor, all series of the proximal phosphorylation cluster were substituted by alanine (S498A, S500A, S501A, S502A, S504A). Similarly, phosphorylatable side chains of serines and threonines within the distal phosphorylation cluster were removed via alanine substitution (S510A, T512A, S513A, T515A) to generate PTH1R-PD2. a NanoBRET assessed recruitment of β-arrestin1 and 2 to the three receptor variants. Briefly, HEK-WT or ΔQ-GRK cells were transfected with either PTH1R-WT, PTH1R-PD1, or PTH1R-PD2 coupled to a C-terminal HaloTag and β-arrestin1- or 2-NanoLuc expression constructs. Upon stimulation with PTH(1-34), the concentration-dependent change in BRET signal was measured. Data are shown as Δ net BRET change in percent and represented as the mean of at least three independent repetitions (PTH1R-WT n = 4; all other conditions n = 3) ± SEM and normalised to PTH1R-WT recruitment in HEK-WT. The…

• 

  Fig. 5. The PTH1R phosphorylation state induces specific conformational rearrangements in β-arrestin1 and 2. a Conformational change of β-arrestin1 and 2 biosensors labelled in the respective N-domain, interacting with PTH1R-WT, PTH1R-PD1, PTH1R-PD2 in HEK-WT, or PTH1R-WT in ΔQ-GRK. In short, HEK-WT or ΔQ-GRK cells were transfected with either an untagged PTH1R-WT, PTH1R-PD1, or PTH1R-PD2 expression construct and one β-arrestin conformational change biosensor, FlAsH-labelled and stimulated with 3 µM PTH(1-34). Conformational change data are shown as Δ net BRET change in percent, mean of at least three independent repetitions (all measurements in ΔQ-GRK n = 4; β-arrestin2-F3 PTH1R-WT, -PD1 n = 4; all other conditions n = 3) + SEM. The statistical significance was calculated by one-way ANOVA, followed by a two-sided Dunnett’s test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). The complete results of the statistical analysis are combined in Supplementary Table 5. b Shows a radar chart representation of the data from a and c. The data for each individual sensor were normalised to the respective conformational change value induced by PTH1R-WT in each dataset. c Shows…

• 

  Fig. 6. Phosphorylation states of the receptor determine PTH1R internalisation and ERK1/2 signalling. a Time-dependent BRET change between a NanoLuc-tagged receptor and CAAX-YFP or FYVE-mNeonGreen (nG), measured for PTH1R-WT, -PD1 and -PD2 in HEK-WT. Results are shown as mean of three independent repetitions (n = 3) and 95% confidence intervals (coloured areas). b schematic depiction of the utilised measurement systems to comprehensively investigate the initial internalisation and trafficking characteristics of the PTH1R. c and d Concentration-dependent BRET changes, of all three investigated PTH1R variants in the CAAX association and FYVE dissociation assays, as measured in HEK-WT, β-arrestin1/2 double knockout and ΔQ-GRK cells. Cells were transfected with CAAX-YFP or FYVE-mNeonGreen and NanoLuc-tagged constructs of either PTH1R-WT, -PD1, or -PD2 and stimulated with varying concentrations of PTH(1-34), as indicated. Results are shown as mean of three independent repetitions (n = 3) ± SEM. e and f Analogous measurements, utilising the energy transfer between NanoLuc-fused β-arrestin constructs in the presence of untagged PTH1R (-variants), in order to assess the endosomal delivery of…

• 

  Fig. 7. Possible geometry of ‘hanging’ and ‘core’ complex configurations between PTH1R and β-arrestin1 and 2. Molecular modelling of differently formed ‘hanging’ and ‘core’ complex configurations between the active and phosphorylated PTH1R (PDB: 6NBF) and β-arrestin1 (PDB: 4JQI) and 2 (PDB: 6K3F), respectively. The results for β-arrestin1 interactions with the PTH1R are depicted in the top row, while the bottom row shows analogous results obtained for β-arrestin2. Our data suggest that β-arrestin1 (top row) interactions with the PTH1R rely more strongly on the distal phosphorylation sites, while the proximal phosphorylation does not seem to stabilise these complexes. Additionally, for β-arrestin1 both ‘hanging’ and ‘core’ PTH1R–β-arrestin1 complex configurations are presumably stabilised by membrane anchorage of the exposed C-edge loops47. Hence, we propose that PTH1R–β-arrestin1 interactions rely on a 2-site interaction in the ‘hanging’ configuration, while the additional engagement of the FLR adds a third interaction site in the ‘core’ configuration. In contrast, for β-arrestin2 the bottom row depicts that the ‘hanging’ PTH1R–β-arrestin2 complex was characterised with a low stability…