Sortase ligation enables homogeneous GPCR phosphorylation to reveal diversity in β-arrestin coupling

Abstract

The ability of G protein-coupled receptors (GPCRs) to initiate complex cascades of cellular signaling is governed by the sequential coupling of three main transducer proteins, G protein, GPCR kinase (GRK), and β-arrestin. Mounting evidence indicates these transducers all have distinct conformational preferences and binding modes. However, interrogating each transducer's mechanism of interaction with GPCRs has been complicated by the interplay of transducer-mediated signaling events. For example, GRK-mediated receptor phosphorylation recruits and induces conformational changes in β-arrestin, which facilitates coupling to the GPCR transmembrane core. Here we compare the allosteric interactions of G proteins and β-arrestins with GPCRs' transmembrane cores by using the enzyme sortase to ligate a synthetic phosphorylated peptide onto the carboxyl terminus of three different receptors. Phosphopeptide ligation onto the β₂-adrenergic receptor (β₂AR) allows stabilization of a high-affinity receptor active state by β-arrestin1, permitting us to define elements in the β₂AR and β-arrestin1 that contribute to the receptor transmembrane core interaction. Interestingly, ligation of the identical phosphopeptide onto the β₂AR, the muscarinic acetylcholine receptor 2 and the μ-opioid receptor reveals that the ability of β-arrestin1 to enhance agonist binding relative to G protein differs substantially among receptors. Furthermore, strong allosteric coupling of β-arrestin1 correlates with its ability to attenuate, or "desensitize," G protein activation in vitro. Sortase ligation thus provides a versatile method to introduce complex, defined phosphorylation patterns into GPCRs, and analogous strategies could be applied to other classes of posttranslationally modified proteins. These homogeneously phosphorylated GPCRs provide an innovative means to systematically study receptor-transducer interactions.

Keywords: G protein-coupled receptor; allostery; phosphorylation; sortase; β-arrestin.

Fig. 1.

Illustration showing the two-step binding mode of β-arrestin. Ligand (L) binding to the extracellular orthosteric binding pocket leads to conformational changes within the GPCR transmembrane region to influence intracellular transducer binding. The phosphorylation (red circles) of the receptor C terminus by GPCR kinase (GRK) initiates the recruitment of β-arrestin (βarr). Conformational changes induced in βarr (βarr*) as a result of binding to the phosphorylated C terminus promotes coupling to the GPCR transmembrane core, which allosterically enhances ligand affinity.

Fig. 2.

Nonphosphorylated β₂AR interacts with Gs heterotrimer but not β-arrestin1. (A) Coomassie-stained gel showing the coimmunoprecipitation of Gs heterotrimer (Gs) or β-arrestin1 (βarr1) with isoproterenol (ISO)-bound FLAG-β₂AR. Loading controls represent 10% of input. (B) Competition binding experiments using radiolabeled [¹²⁵I]-cyanopindolol (CYP). Gs increases ISO affinity for β₂AR HDLs (log IC₅₀: −8.88 ± 0.03) compared with no transducer (log IC₅₀: −6.82 ± 0.03), but βarr1 does not (log IC₅₀: −6.81 ± 0.02). Data shown are the mean of three independent experiments, with error bars representing SE. The green asterisk (*) indicates a log IC₅₀ value significantly different from the control curve (P < 0.05, one-way ANOVA). (C) The fluorescence emission spectrum of bimane-labeled β₂AR HDLs shows a rightward shift and decrease in fluorescence upon addition of ISO, indicative of receptor activation. The effects of ISO are enhanced by Gs but not βarr1. Data shown are representative of three independent experiments.

Fig. 3.

Sortase ligation of a phosphopeptide onto the β₂AR restores its allosteric interaction with β-arrestin1. (A) Cartoon schematic of sortase ligation method. A synthetic phosphopeptide (pp) derived from the vasopressin-2-receptor (V₂R) with three N-terminal glycine residues (GGG-V₂Rpp) is ligated onto receptors containing a C-terminal LPETGGH recognition motif. In the sequence of GGG-V₂Rpp below the schematic, phosphorylated residues are highlighted in red. (B) Coomassie-stained gel showing the coimmunoprecipitation of heterotrimeric Gs and β-arrestin1 (βarr1) with isoproterenol (ISO)-bound, phosphopeptide-ligated FLAG-β₂AR (β₂ARpp). Fab30 binds specifically to V₂Rpp-bound βarr1 (10). Loading controls represent 10% of input. (C and D) Competition binding experiments using radiolabeled [¹²⁵I]-cyanopindolol (CYP) with HDLs containing (C) β₂ARpp or (D) β₂AR ligated to a nonphosphorylated version of the V₂R peptide (β₂ARnp). Gs increases the affinity of ISO for both β₂ARpp and β₂ARnp HDLs (log IC₅₀: −9.15 ± 0.03, −9.02 ± 0.04, respectively) compared with no transducer (log IC₅₀: −6.24 ± 0.04, −6.42 ± 0.09, respectively), but βarr1 only increases ISO affinity for β₂ARpp HDLs (log IC₅₀: −7.14 ± 0.07) and not β₂ARnp HDLs (log IC₅₀: −6.49 ± 0.04). Data shown in C and D are the mean of at least three independent experiments, with error bars representing SE, and asterisks (*) indicate a log IC₅₀ value significantly different from the control curve (P < 0.05, one-way ANOVA). (E) The effects of ISO on the HDL-β₂ARpp-bimane fluorescence emission spectrum are enhanced by Gs and βarr1. Data shown are representative of three independent experiments.

Fig. 4.

The allosteric interaction between phosphorylated β₂AR and β-arrestin1 requires the finger loop of β-arrestin1 but does not require the third intracellular loop of the β₂AR. (A) In competition radioligand binding with β₂ARpp HDLs as described in Fig. 3C, a finger loop deletion mutant of β-arrestin1 (βarr1) (Δ62–77) has minimal effect on isoproterenol (ISO) binding (log IC₅₀s: no transducer, −6.30 ± 0.05; βarr1, −7.25 ± 0.07; βarr1 Δ62–77, −6.48 ± 0.03). (B) βarr1Δ62–77 does not intensify the effects of ISO on the fluorescence spectrum of β₂ARpp-bimane HDLs. Data shown are representative of three independent experiments. (C) In competition radioligand binding with β₂ARpp HDLs containing a deletion of the third intracellular loop (Δ238–267), both Gs (log IC₅₀: −8.85 ± 0.03) and βarr1 (log IC₅₀: −7.63 ± 0.06) retain their ability to increase isoproterenol (ISO) affinity (no transducer, log IC₅₀: −6.91 ± 0.04). Data shown in A and C are the mean of at least three independent experiments, with error bars representing SE, and asterisks (*) indicate a log IC₅₀ value significantly different from the control curve (P < 0.05, one-way ANOVA).

Fig. 5.

The allosteric enhancement of agonist binding induced by β-arrestin1 varies among different receptors. (A) Competition binding experiments with sortase-ligated M₂Rpp HDLs, using [³H]-N-methyl-scopolamine (NMS) as the tracer. Heterotrimeric Gi (100 nM, log IC₅₀: −7.51 ± 0.06) and β-arrestin1 (βarr1) (1 μM, log IC₅₀: −7.06 ± 0.08) increase the affinity of the agonist carbachol to a similar extent (no transducer, log IC₅₀: −5.31 ± 0.09). (B) Competition binding experiments with sortase-ligated MORpp HDLs, using [³H]-naloxone as the tracer. Gi (1 μM, log IC₅₀: −8.22 ± 0.05) increases the affinity of the agonist DAMGO to a far greater extent than βarr1 (1 μM, log IC₅₀: −6.02 ± 0.05) (no transducer, log IC₅₀: −5.71 ± 0.06). Data in A and B are the mean of three independent experiments, with error bars representing SE, and asterisks (*) indicate a log IC₅₀ value significantly different from the control curve (P < 0.05, one-way ANOVA). (C) Comparison of the difference in agonists’ log IC₅₀ values in the presence of their cognate G proteins versus βarr1 for sortase-ligated β₂ARpp (Fig. 3C), M₂Rpp (A), and MORpp (B) HDLs.

Fig. 6.

The extent to which β-arrestin1 engages receptors’ transmembrane cores varies among different receptors. (A) Fluorescence spectra of β-arrestin1 (βarr1) labeled with monobromobimane at residue 70 in the finger loop. Activation of HDL-β₂ARpp by the agonist isoproterenol (ISO) increases βarr1-bimane fluorescence, which is blocked by Nb80 binding to the receptor TM core. Data shown are representative of three independent experiments. (B) Comparison of βarr1-bimane fluorescence by agonist activation of β₂ARpp, M₂Rpp, and MORpp HDLs. The area under the fluorescence emission spectra were determined and normalized to M₂Rpp plus iperoxo (the maximum signal) in each experiment (see Fig. S4 for representative spectra). All three receptors are significantly different from one another (*P < 0.05), and β₂ARpp and M₂Rpp are significantly different from their respective antagonist controls (not indicated, P < 0.05). (C) An in vitro GTPase assay measuring GTP hydrolysis as a readout of G protein activation. The basal level of GTP hydrolysis induced by G protein is robustly increased by HDL-β₂ARpp HDLs in the presence of ISO compared with no ligand (NL) (*P < 0.05), which is blocked (desensitization) by the addition of Nb80 (no significant difference between NL and ISO + Nb80). (D) Inhibition (percentage desensitization) of G protein activation by βarr1 is strongest at M₂Rpp and significantly different from β₂ARpp and MORpp HDLs (*P < 0.05) (see also Fig. S5). Data in B–D are the mean of at least three independent experiments, with error bars representing SE; P values were determined by one-way ANOVA.