GPCR-mediated β-arrestin activation deconvoluted with single-molecule precision

Abstract

β-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that β-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the β-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that β-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for β-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of β-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream β-arrestin-mediated events are directed.

Keywords: G protein-coupled receptor; GPCR; agonist; arrestin; conformational dynamics; phosphorylation; phosphorylation barcode; receptor signaling; single-molecule FRET; β-arrestin.

Figure 1.. β-arrestin1 C-tail dynamic behavior in the basal state.

A, Schematic of βarr binding to an agonist-activated GPCR in the plasma membrane. B, Representative MD frames of simulated βarr1 in the inactive state showing the distal βarr1 tail outside the extended groove. C, MD frames from a subset of these simulations showing the distal tail can dock within the extended groove, bringing the mean dye positions closer together. D, Schematic of the smFRET experiment with fluorophore (green and red circles)-labeled βarr1 tail sensor tethered to an imaging surface. E, Representative single-molecule fluorescence (top; donor in green, acceptor in red) and FRET (bottom; blue) time traces of surface-tethered βarr1 recorded with 100 ms time resolution in the absence of ligands, with a rare excursion out of the basal state to a ~0.89 FRET state.

Figure 2.. Activation of β-arrestin1 by V₂Rpp.

A, Schematic of the smFRET experiment where V₂Rpp (orange with red for phosphorylation sites) binding induces βarr1 tail (cyan) disengagement. B, Representative single-molecule traces with state assignment (bottom, red line);surface-tethered βarr1 tail sensor was sequentially exposed to buffer containing the indicated V₂Rpp concentrations (shaded areas). C, Population smFRET histograms for inactive (red) and active (blue and green) states (top; N, number of traces) and corresponding transition density plots displaying the mean FRET values before (x axis) and after (y axis) each transition (bottom; scale bar, 10⁻³ transitions per bin per second) in the presence of the indicated V₂Rpp concentrations. D, Ensemble average high FRET inactive state occupancy induced by increasing concentrations of various peptides: V₂Rpp (red), V₂Rpp with 0.1 μM Fab30 (green), V₂Rbp (blue), and V₂Rnp (black). Lines are fits to dose-response functions with Hill slope of 1.0 and EC₅₀ of 2.6 μM for V₂Rpp alone and 0.012 μM for V₂Rpp with Fab30, respectively. E, Analysis of V₂Rpp interaction with wildtype βarr1 by isothermal titration calorimetry (ITC). Representative binding isotherm (top) and thermogram (bottom), with the best titration curve fit are shown. Summary of thermodynamic parameters obtained by ITC: binding affinity (K_D = 2.4 ± 0.2 μM), stoichiometry (N = 1.1 ± 0.1 sites), enthalpy (ΔH = −20.3 ± 0.4 kcal/mol), and entropy (−TΔS = 13.9 ± 2.0 kcal/mol). F, Dwell time histograms (symbols) in the inactive (top) and active (bottom) states from experiments corresponding to panels C-D with spline fits (lines). G, Apparent rate constants for V₂Rpp binding (blue) and unbinding (red) of 0.10 μM⁻¹ s⁻¹ and 0.33 s⁻¹, respectively. Error bars, mean ± S.D. of at least two repeats with at least 3,500 traces for each condition and a total of more than 33,000.

Figure 3.. Activation of β-arrestin1 by heparin, IP6 and PIP2.

A, Structure of heparin. B, Representative smFRET traces from experiments imaging βarr1 tail sensor in the presence of 1 μM heparin with 10 ms time resolution. C, Overlay of FRET histograms experiments with 1 μM heparin (purple) and V₂Rpp (red). D, FRET histograms and E, high-FRET state occupancy (symbols) from experiments imaging βarr1 tail sensor in the presence of the indicated concentrations of heparin. Line is a fit to a dose-response function (red line) with Hill slope of 1.0 and EC₅₀ of 17 nM. F, Example trace recorded at 100 ms time resolution in the presence of 1 μM heparin showing rare switching between dynamic modes. Error bars, mean ± S.D. of two repeats. G, Population FRET histograms of βarr1 C tail sensor, imaged at 100 ms time resolution, with indicated concentrations of IP₆ and H, PIP₂. I, Ensemble average high-FRET inactive state occupancy as a function of V₂Rpp concentrations in the absence and presence of indicated concentrations of IP₆ or PIP₂ (symbols) fit to dose-response functions (lines), revealing no substantial change in EC50 values.

Figure 4.. Effect of activating mutations on β-arrestin1 tail disengagement.

A, Basal structure of βarr1 (PDB: 1G4M) showing regions and residues associated with the three-element interaction (left) and finger-loop proximal (right) mutants. B, WT-, C, I386A/V387A/F388A- and D, E313K-βarr1 tail sensor was imaged in the absence and presence of varying concentrations of V₂Rpp. Left, Representative smFRET traces in the absence (left) and presence (right) of V₂Rpp. Middle, population FRET histograms (symbols) with spline fits (lines). Right, ensemble average occupancy in the high-FRET inactive state as a function of V₂Rpp concentration (symbols) fit to dose-response functions (lines) with Hill slope of 1.0 and EC₅₀ values of 2.6(WT, black), 0.030 (3-EI mutant, green), 0.070 (E313K, red), and 0.063 μM (R76A/K77A/D78A, blue). Error bars, mean ± S.D. of at least two repeats.

Figure 5.. Activation of β-arrestin1 by phosphorylated, agonist-bound receptor.

A, Schematic of the experiment showing βarr1 tethered to an imaging surface interacting with pβ₂V₂R free in solution and bound to ligands of varying efficacy. B, Representative FRET (blue) and state assignment (red) traces and C, population FRET histograms from smFRET imaging of βarr tail sensor conducted in the presence of 1 μM pβ₂V₂R and 10 μM carazolol, no ligands, 200 μM epi, 3 μM epi, or 3 μM epi with 10 μM Cmpd-6FA, respectively. D, High-FRET inactive state occupancy as a function of pβ₂V₂R concentration in the presence of saturating concentrations of the indicated receptor-binding ligand (symbols) fit to dose response functions (lines) with a Hill slope of 1 and EC50 values of 44 (carazolol; red), 30 (apo; gray), and 1.0 μM (epi; green). E, High-FRET inactive state occupancy from experiments conducted in the presence of 1 μM pβ₂V₂R and the indicated concentration of epinephrine in the absence (blue) and presence (green) of 10 μM Cmpd-6FA fit to dose response functions with Hill slope of 1.0 and EC50 values of 3.7 and 1.0 μM, respectively. F-H, Histograms of dwell times in the mid-FRET active (left) and high-FRET inactive (right) states: F, from experiments corresponding to panels B-C, G, from experiments with 1 μM pβ₂V₂R and the indicated concentrations of epinephrine, and H, from experiments with 1 μM V₂Rpp duplicated from Figure 2 for comparison. I, Representative MD frames of the simulated pβ₂V₂R-βarr complex model bound to epinephrine (cyan) and Cmpd-6FA (magenta) showing the positions and dynamics of P1 to P8 in V₂Rpp ( wheat color). Error bars, mean ± S.D. of two repeats.

Figure 6.. The β₂AR C-tail inhibits β-arrestin recruitment in living cells.

A, Schematic of the bystander BRET-based βarr membrane recruitment assay performed in living cells where the N-terminus of βarr is fused to the donor Rluc8 and a plasma membrane marker is fused to the acceptor citrine. Upon agonist stimulation, receptors with unmodified C-tails recruit βarr to the plasma membrane, resulting in an increase of BRET between the donor and acceptor molecules. B, Dose-response curves of βarr2 recruitment to the plasma membrane by wildtype (WT) β₂AR (black squares) and β₂AR-365tr (blue triangles) in HEK293 GRK2/3/5/6 knockout cells in the absence (GRK KO) or D, presence of GRK2 expression via transient transfection (GRK2 rescue). Dose-response curves of three independent experiments performed with triplicate samples (mean ± S. E.). C, βarr2 membrane recruitment by WT β₂AR and β₂AR-365tr in response to10 μM isoproterenol stimulation in the absence and E, presence of GRK2 transfection. Bars represents the mean ± S. E. of three independent experiments performed in triplicate. Symbols represent the measured drug-induced BRET value. **, p = 0.0056; ***, p = 0.0002, unpaired, two-tailed Mann-Whitney test.