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. 2012:3:995.
doi: 10.1038/ncomms2000.

Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin

Martha E Sommer  1 , Klaus Peter Hofmann, Martin Heck
Affiliations
Free PMC article

Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin

Martha E Sommer et al. Nat Commun. 2012.
Free PMC article

Erratum in

  • Nat Commun. 2012;3:1273

Abstract

G-protein-coupled receptors are universally regulated by arrestin binding. Here we show that rod arrestin induces uptake of the agonist all-trans-retinal [corrected] in only half the population of phosphorylated opsin in the native membrane. Agonist uptake blocks subsequent entry of the inverse agonist 11-cis-retinal (that is, regeneration of rhodopsin), but regeneration is not blocked in the other half of aporeceptors. Environmentally sensitive fluorophores attached to arrestin reported that conformational changes in loop(V-VI) (N-domain) are coupled to the entry of agonist, while loop(XVIII-XIX) (C-domain) engages the aporeceptor even before agonist is added. The data are most consistent with a model in which each domain of arrestin engages its own aporeceptor, and the different binding preferences of the domains lead to asymmetric ligand binding by the aporeceptors. Such a mechanism would protect the rod cell in bright light by concurrently sequestering toxic all-trans-retinal [corrected] and allowing regeneration with 11-cis-retinal.

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Figures

Figure 1
Figure 1. Functional test of receptor phosphorylation.
Arrestin was titrated against 10 μM RhoP (pH 8, 2 °C), and the amount of stabilized Meta II following an activating flash (20%) was measured as shown in the inset. A fit of equation (3) to the data indicated that one arrestin bound for every light-activated RhoP with an apparent Kd of 27 nM, which corresponds to a true Kd of 2.2 nM when corrected for the Meta I/Meta II equilibrium. Inset—binding to and stabilisation of Meta II over its precursor Meta I results in an increase in absorbance (380–417 nm). In the control experiment (black trace), 300 μM Gtα-peptide (the high-affinity analogue peptide derived from the C-terminus of the α-subunit of transducin, VLEDLKSCGLF) stabilized 100% of receptors as Meta II. The coloured traces correspond to the arrestin titration. Experiments were carried out as described previously.
Figure 2
Figure 2. Arrestin reforms Meta II-P from OpsP and ATR.
(a) Arrestin was titrated against 4 μM light-induced Meta II-P (black symbols), 4 μM OpsP+40 μM ATR (white symbols) or 4 μM OpsP (grey symbols) in isotonic buffer, and arrestin binding was measured by pull down. Curves were fit as described in the Methods, and apparent Kd and stoichiometry values are listed in Supplementary Table S1. (b) OpsP (4 μM) and ATR (4 μM) were mixed together with or without arrestin (4 μM) in 50 mM HEPES buffer pH 7 without salt. After equilibration, 20 mM t-bHA was added to remove peripheral Schiff bases, followed by acid denaturation and detergent solubilisation. The resulting absorbance spectra of the samples are shown. Inset—difference spectrum calculated by subtracting the spectrum without arrestin from that with arrestin. (c) The time course of regeneration of OpsP (4 μM) by 11-cis-retinal (20 μM) was followed by monitoring the absorbance at 500 nm (arrestin: 4 μM; ATR: 40 μM; 50 mM HEPES buffer pH 7 without salt, 20 °C).
Figure 3
Figure 3. Arrestin and retinoid titrations.
(a) ATR was titrated against 4 μM arrestin+4 μM OpsP. (b) Arrestin was titrated against 4 μM OpsP+40 μM ATR. Arrestin binding (red), retinal Schiff base formation (blue) and blocked rhodopsin regeneration (green) were measured as described in the text. All experiments were performed in HEPES buffer pH 7 without salt, except for the pull-down experiment in (a) (red), where isotonic buffer was necessary to reduce agonist-independent arrestin binding. For the regeneration data (green), the fitted curves intercept the y axis at ~0.5, reflecting the fact that not all OpsP was regenerated due to experimental constraints (Methods). Both single-site (solid traces) and two-site (dashed traces) binding models were fit to the retinal titration data. (c) ATR was titrated against 4 μM arrestin+4 μM OpsP using an OpsP preparation in which ~70% of receptors were able to bind arrestin as light-induced Meta II-P. The fitted curve reports an apparent Kd of 3.4 μM and a stoichiometry of one arrestin per 4 receptors. Inset—time course of regeneration of OpsP (4 μM) with 11-cis-retinal (20 μM) in the absence or presence of arrestin (4 μM) and ATR (40 μM). (d) Arrestin titrations using mutants A366NBD (black symbols) or S344NBD (grey symbols) were performed against 4 μM Meta II-P or 4 μM OpsP+40 μM ATR using the same under-phosphorylated receptors as in (c). The fitted curves report that that one arrestin bound for every 1.8 Meta II-P or 3.6 μM OpsP/ATR (200 nM<Kd<300 nM). For both (c) and (d), arrestin binding in isotonic buffer was measured by pull down. (e) ATR and beta-ionone were titrated against 4 μM arrestin+4 μM OpsP (100% phosphorylated) in isotonic buffer, and arrestin binding was measured by pull down. The ATR data and fits from panel (a) are shown for reference. Note that beta-ionone caused scattering artefacts at higher concentrations. For all panels, data points from independent experiments are represented by differently shaped symbols, and the combined data points were used to fit the binding curves described in the Methods.
Figure 4
Figure 4. Physiological context of ATR trapping.
(a) Isolated ROS membranes containing 30 μM RhoP (upper panel) or 300 μM RhoP (lower panel) were fully photoactivated without arrestin (closed symbols) or with arrestin (open symbols), which was present at the physiologically relevant ratio of 0.67 arrestin per rhodopsin. Meta II-P decay was monitored as the decrease in t-bHA-resistant retinal Schiff base. Each experiment was performed twice in isotonic buffer at 37 °C. (b) A sample containing 4 μM OpsP+20 μM ATR, with (triangles) or without (circles) 4 μM arrestin was monitored for regeneration after the addition of 11-cis-retinal (12 μM). NADPH (200 μM), the essential cofactor of RDH, was added 60 min after the start of regeneration to an arrestin-containing sample (open triangles). Experiments were performed in 50 mM HEPES buffer pH 7 without salt, 20 °C. (c) The fluorescence of arrestin I72NBD (2 μM, blue trace) in the presence of OpsP (2 μM) was monitored over time after subsequent additions of ATR (4 μM, t=200 s), NADPH (200 μM, t=400 s) and NH2OH (10 mM, t=1600 s). ATR formation after addition of NADPH to samples of OpsP (2 μM) and ATR (4 μM) was monitored in the absence (dark red) or presence of 2 μM unlabelled arrestin (red). Experiments were performed in 50 mM HEPES buffer pH 7 without salt, 35 °C.
Figure 5
Figure 5. Site-directed fluorescence experiments.
(a) Location of single cysteine substitutions and attachment of the NBD fluorophore on a model of arrestin derived from the crystal structure (PDB code 1CF1). The N-domain is coloured blue, the C-domain is green and the C-terminus is grey. (b) For each panel, 1 μM fluorescently labelled arrestin was mixed with a fourfold excess of dark-state RhoP. Emission spectra were recorded before (red traces) and after (blue traces) a 10-s illumination (>495 nm). Spectra were background-subtracted and normalised.
Figure 6
Figure 6. Receptor titrations.
(a) Dark-state RhoP was titrated against 2 μM arrestin I72NBD. (b) Light-induced Meta II-P was titrated against 2 μM arrestin I72NBD. (c) OpsP was titrated against 2 μM arrestin I72NBD. (d) OpsP plus a 10-fold molar excess of ATR was titrated against 2 μM arrestin I72NBD. (e) Dark-state RhoP was titrated against 2 μM arrestin S344NBD. (f) Light-induced Meta II-P was titrated against 2 μM arrestin S344NBD. (g) OpsP was titrated against 2 μM arrestin S344NBD. (h) OpsP plus a 10-fold molar excess of ATR was titrated against 2 μM arrestin S344NBD. Arrestin binding was measured by pull down (closed symbols), and the steady-state fluorescence was measured in parallel on identical samples (open symbols). In the case of the OpsP/ATR titrations, 20 mM t-bHA was added before fluorescence measurements to remove nonspecific retinal Schiff bases. The fluorescence is expressed as the fractional increase over that of unbound arrestin. For each panel, the left axis refers to arrestin pulled down, and the right axis refers to fluorescence. Each point represents the average of two independent experiments, which were performed in 50 mM HEPES buffer pH 7.
Figure 7
Figure 7. Model of different binding modes of arrestin.
(a) Crystal structure of Meta II (M II) (PDB code 3PXO). (b) Rhodopsin (Rho) (1U19), which is similar to inactive opsin in conformation. (c) Arrestin with closed conformation of loop-72 (molecule D from 1CF1). (d) Arrestin with extended conformation of loop-72 (molecule A from 1CF1). The N-domain is coloured blue, the C-domain is coloured green and the C-terminus has been omitted for clarity. Sites labelled in this study are indicated. (e) As described in the text, arrestin binds light-induced Meta II-P in 1:1 or 1:2 complexes. (f) Arrestin binds functional dimers of OpsP, in which agonist (ATR) can enter one protomer and thereby reform Meta II-P. Meta II-P decay results in an equilibrium of free and bound ATR.
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References

    1. Park J. H., Scheerer P., Hofmann K. P., Choe H. W. & Ernst O. P. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008). - PubMed
    1. Scheerer P. et al.. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008). - PubMed
    1. Hofmann K. P. et al.. A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem. Sci. 34, 540–552 (2009). - PubMed
    1. Choe H. W. et al.. Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011). - PubMed
    1. Rasmussen S. G. et al.. Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011). - PMC - PubMed

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