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The conformational signature of β-arrestin2 predicts its trafficking and signalling functions


Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling1,2. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits3,4. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based ‘signalsomes’5,6. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters7 to monitor conformational changes in β-arrestin2, here we show that GPCRs impose distinctive arrestin ‘conformational signatures’ that reflect the stability of the receptor–arrestin complex and role of β-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in β-arrestin2 conformation. Our findings demonstrate that information about ligand–receptor conformation is encoded within the population average β-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands8,9 and in identifying factors that dictate arrestin conformation and function.

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Figure 1: Design and characterization of rLuc–β-arrestin2–FlAsH BRET reporters.
Figure 2: Relationship between GPCR–β-arrestin2 complex formation, rLuc–β-arrestin2–FlAsH BRET signature, and arrestin-dependent ERK1/2 activation for six different GPCRs.
Figure 3: Effect of GPCR–arrestin trafficking pattern and ligand structure on the rLuc–β-arrestin2–FlAsH BRET conformational signature.


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This work was supported by National Institutes of Health grants DK055524 (L.M.L.) and GM095497 (L.M.L.), funds provided by Dialysis Clinics, Inc. (T.A.M), and the Research Service of the Charleston, SC Veterans Affairs Medical Center (L.M.L.). Supported by Canadian Institutes of Health Research operating grant MOP-74603 (S.A.L.). National Institutes of Health grant RR027777 (L.M.L.) supported the FLIPRTETRA facility. The contents of this article do not represent the views of the Department of Veterans Affairs or the United States Government.

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Authors and Affiliations



M.-H.L., K.A.M., E.G.S., J.Y.K, and S.A.L. performed experimental measurements and data analysis. T.A.M., Y.K.P., and S.A.L. provided technical expertise. M.-H.L. and L.M.L. conceived the project. All authors contributed to preparation of the manuscript and approved the final version.

Corresponding author

Correspondence to Louis M. Luttrell.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Time-course and relationship of the β-arrestin2 intramolecular FlAsH BRET signal to receptor occupancy.

a, Time-course of AT1AR-induced changes in intramolecular FlAsH BRET. HEK293 cells were co-transfected with plasmid cDNA encoding AT1AR and the indicated rLuc–β-arrestin2–FlAsH reporter. Stimulations were carried out at a saturating concentration of AngII for the indicated times. The graph depicts the mean ± s.e.m. of independent biological replicates of ligand-induced Δnet BRET for each rLuc–β-arrestin2–FlAsH construct (n = 6). b, Ligand concentration dependence of PTH1R- and α1BAR-induced changes in intramolecular FlAsH BRET. HEK293 cells were co-transfected with plasmid cDNA encoding the PTH1R or α1BAR and the rLuc–β-arrestin2–FlAsH5 reporter. Stimulations were for 2 min using the indicated agonist concentration. The graph depicts the mean ± s.e.m. of independent biological replicates of ligand-induced Δnet BRET (n = 5). The EC50 for PTH(1–34) (PTH1R) and phenylephrine (α1BAR) were 30 nM and 80 nM, respectively. In all panels: *P < 0.05, #P < 0.005, greater or less than vehicle stimulated control.

Extended Data Figure 2 G-protein-coupling profiles of selected GPCRs.

a, Representative time-courses of cAMP luminescence following stimulation of HEK293 GloSensor cAMP cells transfected with each of six GPCRs. For the Gi/o-coupled S1P1R and α2AAR, stimulations were carried out in the presence of 10 μM forskolin to detect inhibition of adenylyl cyclase. Each panel depicts the agonist effect (green) compared to the control response to 10 μM forskolin (grey) measured in adjacent wells. Data are presented in relative luminescence units (RLU). b, Representative time-courses of intracellular calcium fluorescence following stimulation of HEK293 cells transfected with the same panel of GPCRs. Each panel depicts the agonist effect (blue) compared to the control response to the calcium ionophore A23187 (lavender) measured in adjacent wells. Data are presented in relative fluorescence units (RFU).

Extended Data Figure 3 Pertussis toxin sensitivity of ERK1/2 activation by Gi/o-coupled GPCRs.

HEK293 cells transfected with the β2AR, S1PR1 or α2AAR were serum-deprived overnight in the presence or absence of 1 ng ml−1 Bordetella pertussis toxin (PTX) before 5 min stimulation with isoproterenol, S1P or UK14303, respectively. Representative phospho-ERK1/2 immunoblots are shown above bar graphs depicting the mean ± s.e.m. of independent biological replicates (n = 5, β2AR, S1P1R and α2AAR). Responses were normalized to the basal level of phospho-ERK1/2 in non-stimulated samples. *P < 0.05, #P < 0.005, less than stimulated response in the absence of pertussis toxin.

Extended Data Figure 4 Concentration-response relationship between FlAsH5 signal and arrestin-dependent ERK1/2 activation.

a, Relationship between α1BAR-induced change in FlAsH5 Δnet BRET and arrestin-dependent ERK1/2 activation at varying agonist concentration. The percent maximal phenylephrine-induced FlAsH5 Δnet BRET was determined in HEK293 cells transfected with α1BAR and rLuc–β-arrestin2–FlAsH5 expression plasmids (left). The concentration dependence of phenylephrine-stimulated ERK1/2 activation was determined in α1BAR-expressing HEK293 FRT/TO β-arrestin1/2 shRNA cells stimulated for 5 min (centre). β-arrestin1/2-dependent ERK1/2 activation was defined as the fold difference between agonist-stimulated ERK1/2 phosphorylation in the absence (total ERK1/2 signal) and presence (β-arrestin1/2-independent ERK1/2 signal) of doxycycline. A representative phospho-ERK1/2 immunoblot is shown above a graph depicting the mean ± s.e.m. of independent biological replicates (n = 4). EC50 for total ERK1/2, β-arrestin1/2-independent ERK1/2, and β-arrestin1/2-dependent ERK1/2 were 64 nM, 27 nM and 334 nM, respectively. Right, the relationship between percent maximal α1BAR-induced change in FlAsH5 Δnet BRET and β-arrestin1/2-dependent ERK1/2 activation over a range of agonist concentrations. In all panels, *P < 0.05, #P < 0.005, greater than nonstimulated. b, Relationship between GPCR-induced change in FlAsH5 Δnet BRET and arrestin-dependent ERK1/2 activation at saturating agonist concentration. The ligand-induced FlAsH5 Δnet BRET was determined in HEK293 cells transfected with the indicated GPCR and rLuc–β-arrestin2–FlAsH5 expression plasmids. The graph depicts the mean ± s.e.m. of independent biological replicates (n = 5).

Extended Data Table 1 G-protein-coupling and trafficking profiles of selected GPCRs
Extended Data Table 2 Primer sequences used to generate rLuc–β-arrestin2–FlAsH1–6

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Lee, MH., Appleton, K., Strungs, E. et al. The conformational signature of β-arrestin2 predicts its trafficking and signalling functions. Nature 531, 665–668 (2016).

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