I8-arachnotocin–an arthropod-derived G protein-biased ligand of the human vasopressin V2 receptor
Leopold DuerrauerEdin MuratspahićJasmin GattringerPeter KeovHelen C. MendelKevin D. G. PflegerMarkus MuttenthalerChristian W. Gruber
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Abstract The neuropeptides oxytocin (OT) and vasopressin (VP) and their G protein-coupled receptors OTR, V 1a R, V 1b R, and V 2 R form an important and widely-distributed neuroendocrine signaling system. In mammals, this signaling system regulates water homeostasis, blood pressure, reproduction, as well as social behaviors such as pair bonding, trust and aggression. There exists high demand for ligands with differing pharmacological profiles to study the physiological and pathological functions of the individual receptor subtypes. Here, we present the pharmacological characterization of an arthropod ( Metaseiulus occidentalis ) OT/VP-like nonapeptide across the human OT/VP receptors. I8-arachnotocin is a full agonist with respect to second messenger signaling at human V 2 R (EC 50 34 nM) and V 1b R (EC 50 1.2 µM), a partial agonist at OTR (EC 50 790 nM), and a competitive antagonist at V 1a R [pA 2 6.25 (558 nM)]. Intriguingly, I8-arachnotocin activated the Gα s pathway of V 2 R without recruiting either β-arrestin-1 or β-arrestin-2. I8-arachnotocin might thus be a novel pharmacological tool to study the (patho)physiological relevance of β-arrestin-1 or -2 recruitment to the V 2 R. These findings furthermore highlight arthropods as a novel, vast and untapped source for the discovery of novel pharmacological probes and potential drug leads targeting neurohormone receptors.Keywords:
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β-Arrestins are critical regulators of G protein-coupled receptors (GPCRs) that desensitize G protein signaling, promote receptor internalization, and initiate signaling on their own. Recent structural findings indicate that β-arrestins adopt different conformations upon interaction with agonist-activated GPCRs. Here, we established a β-arrestin-2 conformational bioluminescence resonance energy transfer (BRET) sensor composed of the bright Nanoluc BRET donor and the red-shifted CyOFP1 BRET acceptor. The sensor monitors early intramolecular conformational changes of β-arrestin-2 in complex with a wide panel of different class A and class B GPCRs upon agonist activation and with orphan GPCRs known to spontaneously recruit β-arrestin-2. The introduction of the R170E mutant in the β-arrestin-2 sensor allowed the detection of a partially active β-arrestin-2 conformation, which is not dependent on receptor phosphorylation, while the deletion of the β-arrestin-2 finger-loop region detected the "tail-conformation" corresponding to the interaction of β-arrestin with the carboxyl-terminal domain of GPCRs. The new sensors combine the advantages of the BRET technique in terms of sensitivity, robustness, and suitability for real-time measurements with a high responsiveness toward early conformational changes to help to elucidate the different conformational states of β-arrestins associated with GPCR activation in living cells.
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β-Arrestins are ubiquitously expressed in all cell types, and function in the desensitization of G- protein coupled receptors (GPCRs), the control of GPCR intracellular trafficking, and the activation of GPCRs to multiple signaling pathways (1-4). Therefore, β-arrestin-mediated signaling constitutes an important part of GPCR signaling in addition to G protein-mediated signaling. As many GPCRs are found to recruit β-arrestin, the β-arrestin recruitment assay has found important use in drug discovery, especially in the discovery of ligands for orphan GPCRs and in situations where the second messenger signaling is unknown (5,6). Furthermore, the discovery of biased GPCR ligands and the findings that distinct G-proteins versus β-arrestin signaling preferences may offer therapeutic advantages over conventional ligands imply that a screening campaign should be designed to focus on the most disease relevant pathways (7-10). In this aspect, the β-arrestin recruitment assay has added an important piece to the repertoire of assay tools in drug discovery.There are four major in vitro assay technologies available on the market that are capable of measuring ligand-induced β-arrestin recruitment: PathHunter β-arrestin Assay (DiscoverX) (11), Tango GPCR Assay (Thermo Fisher Scientific) (12), LinkLight GPCR/ β-arrestin Signaling Pathway Assay (BioInvenu) (13), and Transfluor Assay (Molecular Devices) (14). The PathHunter β-arrestin Assay, Tango GPCR Assay System and LinkLight GPCR/ β-arrestin Signaling Pathway Assays are homogenous, high throughput assays while the Transfluor Assay is a fluorescence image-based assay. All four assays involve the expression of the β-arrestin as a fusion protein with another protein or fragment, while the PathHunter, Tango and LinkLight assays require fusion of the GPCR to another peptide or protein moiety as well. Table 1 compares the principles of these technologies as well as their advantages and limitations.This guideline uses PathHunter β-arrestin from DiscoverX to illustrate the concepts for performing GPCR β-arrestin recruitment assay. The principle behind this guideline can be applied to all the β-arrestin assay technologies.
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Numerous congenital disorders, ranging from night blindness to cancer, are associated with signaling by overactive GPCR mutants. Excessive GPCR signaling also underlies certain acquired pathological conditions. The coupling of most GPCRs to their cognate G proteins is stopped by the natural desensitization mechanism, which includes receptor phosphorylation by GRKs followed by arrestin binding to active phosphoreceptor. Elucidation of the molecular mechanisms of arrestin binding to GPCRs enabled the construction of enhanced arrestins that bind active receptors with higher affinity and even interact with unphosphorylated GPCRs. These mutants have therapeutic potential due to their ability to compensate, reducing excessive activity of mutant and normal GPCRs. The feasibility of this compensational approach was demonstrated in rod photoreceptors predominantly expressing only one receptor, rhodopsin, and visual arrestin-1. Mammals have two nonvisual arrestins, which are fairly promiscuous, interacting with hundreds of GPCR subtypes. Identification of arrestin elements responsible for their receptor preference enables the construction of nonvisual arrestins specifically targeting particular receptors. This allows the construction of enhanced receptor-specific mutants for targeted suppression of signaling by one GPCR, but not others expressed by the same cell. Modification of elements responsible for the interactions with non-receptor partners creates signaling-biased arrestins that direct the signaling to the pathways of choice, further expanding potential therapeutic uses of "designer" arrestins.
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Another way for GPCRs to signal G protein–coupled receptors (GPCRs) normally transmit signals by coupling to heterotrimeric guanine nucleotide–binding proteins (G proteins) or by binding β-arrestin proteins. Smith et al. provide evidence for another mechanism, an approximate combination of the two. They monitored the interaction of vasopressin type 2 receptors (V2Rs) and G α proteins in cultured cells using bioluminescent resonance energy transfer. Even though V2Rs do not signal canonically through G α i proteins, they promoted the formation of complexes containing β-arrestin and G α i , and this led to downstream signaling to extracellular signal-regulated kinase protein kinases. Science , this issue p. eaay1833
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The interaction of arrestins with G-protein coupled receptors (GPCRs) desensitizes agonist-dependent receptor responses and often leads to receptor internalization. GPCRs that internalize without arrestin have been classified as "class A" GPCRs whereas "class B" GPCRs co-internalize with arrestin into endosomes. The interaction of arrestins with GPCRs requires both agonist activation and receptor phosphorylation. Here, we ask the question whether agonists with very slow off-rates can cause the formation of particularly stable receptor-arrestin complexes.The stability of GPCR-arrestin-3 complexes at two class A GPCRs, the β2 -adrenoceptor and the μ opioid receptor, was assessed using two different techniques, fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) employing several ligands with very different off-rates. Arrestin trafficking was determined by confocal microscopy.Upon agonist washout, GPCR-arrestin-3 complexes showed markedly different dissociation rates in single-cell FRET experiments. In FRAP experiments, however, all full agonists led to the formation of receptor-arrestin complexes of identical stability whereas the complex between the μ receptor and arrestin-3 induced by the partial agonist morphine was less stable. Agonists with very slow off-rates could not mediate the co-internalization of arrestin-3 with class A GPCRs into endosomes.Agonist off-rates do not affect the stability of GPCR-arrestin complexes but phosphorylation patterns do. Our results imply that orthosteric agonists are not able to pharmacologically convert class A into class B GPCRs.
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Article Figures and data Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract In addition to their role in desensitization and internalization of G protein-coupled receptors (GPCRs), β-arrestins are essential scaffolds linking GPCRs to Erk1/2 signaling. However, their role in GPCR-operated Erk1/2 activation differs between GPCRs and the underlying mechanism remains poorly characterized. Here, we show that activation of serotonin 5-HT2C receptors, which engage Erk1/2 pathway via a β-arrestin-dependent mechanism, promotes MEK-dependent β-arrestin2 phosphorylation at Thr383, a necessary step for Erk recruitment to the receptor/β-arrestin complex and Erk activation. Likewise, Thr383 phosphorylation is involved in β-arrestin-dependent Erk1/2 stimulation elicited by other GPCRs such as β2-adrenergic, FSH and CXCR4 receptors, but does not affect the β-arrestin-independent Erk1/2 activation by 5-HT4 receptor. Collectively, these data show that β-arrestin2 phosphorylation at Thr383 underlies β-arrestin-dependent Erk1/2 activation by GPCRs. https://doi.org/10.7554/eLife.23777.001 Introduction Arrestins were initially named on the basis of their ability to turn-off the coupling of G protein-coupled receptors (GPCRs) to G proteins and thereby inhibit G protein-dependent GPCR signaling. Over the last decade, it has become evident that β-arrestins are also important signal transducers and that a number of biological functions exerted by GPCRs are mediated by β-arrestin-dependent signaling (Shenoy and Lefkowitz, 2011; Lefkowitz and Shenoy, 2005; Latapy and Beaulieu, 2013; Beaulieu et al., 2007). β-arrestins act as multifunctional scaffolds that recruit multiple signaling molecules, such as the core Extracellular signal-regulated kinase Erk1/2 signaling module, composed of Raf1, MAP kinase kinase MEK1 and MAP kinases Erk1/2, a process leading to Erk1/2 activation (Reiter and Lefkowitz, 2006; DeFea et al., 2000; Luttrell et al., 2001). Typically, most GPCRs induce temporally distinct and spatially segregated G protein-dependent and β-arrestin-dependent activation of Erk: G protein-mediated Erk activation is rapid and transient, reaching a maximal level within a few minutes and is followed by translocation of activated Erk into the nucleus to promote gene transcription and cell proliferation. In contrast, β-arrestin-mediated Erk1/2 activation is slower in onset, requiring 5–10 min to reach maximal level, persists more than 1 hr and mainly occurs in the cytosol (Reiter and Lefkowitz, 2006; Ahn et al., 2004a; Shenoy et al., 2006). However, Erk1/2 signaling elicited by certain GPCRs does not fit this classic bimodal activation pattern: for example, the activation of Erk1/2 pathway by 5HT2C receptors is entirely dependent on β-arrestins (Labasque et al., 2008), whereas the engagement of this pathway by the 5-HT4 receptor does not require β-arrestin recruitment (Barthet et al., 2007). In addition, some GPCRs such as µ-opioid receptor induce β-arrestin-dependent activation of Erk1/2 that translocate to the nucleus (Zheng et al., 2008). Accumulating evidence indicates that β-arrestin recruitment to GPCRs and engagement of β-arrestin-mediated signaling depend on both receptor conformational state and a complex pattern of receptor phosphorylation elicited by GPCR kinases (GRKs) (Reiter and Lefkowitz, 2006; Shukla et al., 2011). This phosphorylation barcode is translated into specific β-arrestin conformations that dictate selective signaling and the nature of β-arrestin intracellular functions. Another mechanism that might underlie β-arrestin dependency of receptor-operated signaling is the phosphorylation of β-arrestins themselves. Several phosphorylated residues have been identified in the β-arrestin sequences, including Ser412 for β-arrestin1 (Lin et al., 1997; Barthet et al., 2009; Lin et al., 1999) and Thr276, Ser361 and Thr383 for β-arrestin2 (Lin et al., 2002; Paradis et al., 2015; Kim et al., 2002). These phosphorylation events affect GPCR internalization and/or sequestration and, consequently, steady-state level of GPCR cell-surface expression: phosphorylation of β-arrestin1 at Ser412 by Erk1/2 as well as the phosphorylation of β-arrestin2 at both Ser361 and Thr383 reduce their ability to induce internalization of β2-adrenergic receptor (Lin et al., 1997, 1999, 2002), whereas phosphorylation of β-arrestin2 at Ser14 and Thr276 promotes intracellular sequestration of CXCR4 receptor (Paradis et al., 2015). These results indicate that β-arrestin2 phosphorylation exerts contrasting effects on GPCR trafficking that may depend on the nature of the phosphorylated residue(s) and of the GPCR. Another phosphorylated site (Ser178) was identified in rat/mouse β-arrestin2 and its phosphorylation state affects endosomal trafficking of various GPCRs (Khoury et al., 2014). However, this serine residue is not conserved in other species including human, suggesting different regulations of endosomal GPCR trafficking between species. Additional phosphorylated residues have been found on β-arrestin2 in large-scale phosphoproteomics screens, but their functional relevance remains to be established (Pighi et al., 2011; Sharma et al., 2014; Ballif et al., 2008; Villén et al., 2007; Choudhary et al., 2009; Jørgensen et al., 2009). In contrast to its role in GPCR trafficking, the influence of β-arrestin phosphorylation on GPCR-operated β-arrestin-dependent signaling such as Erk1/2 activation remains unexplored. Here, we investigated the impact on β-arrestin1 and 2 phosphorylation of expression/stimulation of 5-HT2C and 5-HT4 receptors, two GPCRs that differ in their β-arrestin dependency to promote Erk1/2 activation, using high-resolution mass spectrometry. We identified several previously described as well as novel phosphorylated residues on β-arrestin2, while only one phosphorylated site was found on β-arrestin1. Of these, only phosphorylation of β-arrestin2 at Thr383 exhibited a strong increase upon 5-HT2C receptor stimulation. Furthermore, Thr383 was poorly phosphorylated in cells expressing 5-HT4 receptor and its phosphorylation was only slightly enhanced by agonist treatment. These findings prompted functional studies to evaluate the influence of this phosphorylation event in Erk1/2 phosphorylation elicited by stimulation of 5-HT2C receptor and other GPCRs known to engage Erk signaling in a β-arrestin-dependent manner, in comparison with 5-HT4 receptor stimulation, which induces Erk1/2 activation through a β-arrestin-independent mechanism. Results 5-HT2C and 5-HT4 receptor stimulation induce distinct patterns of β-arrestin phosphorylation To characterize in a global manner the impact of 5-HT2C and 5-HT4 receptor expression/stimulation on the phosphorylation state of β-arrestins, YFP-tagged versions of β-arrestin1 or β-arrestin2 were co-expressed with Myc-tagged 5-HT2C or 5-HT4 receptors in HEK-293 cells. Cells were then treated with vehicle or 5-HT for 5 or 30 min. β-arrestins were immunoprecipitated using GFP nanobodies coupled to sepharose beads (GFP-Trap), resolved by SDS-PAGE, detected by colloidal Coomassie blue staining (Figure 1—figure supplement 1) and digested in-gel with trypsin. Analysis of the resulting peptides by nano-LC-MS/MS yielded 88% and 85% sequence coverage for β-arrestin1 and β-arrestin2, respectively, with a p-value threshold of 0.01 for peptide identification (Figure 1—figure supplement 2). Only one phosphorylated site (Thr374) was identified on β-arrestin1, but the corresponding phosphorylated peptide was only detected in cells expressing 5-HT4 receptor, and with a low phosphorylation index, as estimated by dividing the MS signal intensity of the phosphorylated peptide by the sum of MS signal intensities of the phosphorylated and the corresponding non-phosphorylated peptide (Figure 1—source data 1, see also the fragmentation spectrum that pinpoints the position of the phosphorylation site on Figure 1—figure supplement 3). Moreover, label-free quantification of the MS signal of this phosphorylated peptide in cells treated or not with 5-HT indicated that Thr374 phosphorylation is not affected by 5-HT exposure (Figure 1—source data 1). Previous studies using a phosphorylated site-specific antibody have identified Ser412 as an additional phosphorylated residue on β-arrestin1 (Lin et al., 1997; Barthet et al., 2009; Lin et al., 1999). They also indicated that Ser412 phosphorylation state is modified upon activation of various GPCRs, including the 5-HT4 receptor (Barthet et al., 2009). Our MS/MS analyses did not identify this phosphorylated residue, but a low-intensity ion signal corresponding to the theoretical m/z of a peptide comprising the phosphorylated Ser412 was detected in cells expressing the 5-HT4 receptor and exposed for 30 min to 5-HT. Six phosphorylated residues (Thr178, Ser194, Ser267/268, Ser281, Ser361 and Thr383) were identified on β-arrestin2 (Figure 1A, Figure 1—source data 1 and Figure 1—figure supplements 3–5). Note that the fragmentation spectrum of the CPVAQLEQDDQVSPp(S267S268)TFCK peptide did not provide enough information to discriminate between a phosphorylation at Ser267 or at Ser268, but the precursor peptide m/z clearly showed that it carries only one phosphorylated residue (Figure 1—figure supplement 3). Relative quantification of MS signals of β-arrestin2 phosphorylated peptides showed that the phosphorylation of these residues except for Thr383 was not affected by 5-HT2C or 5HT4 receptor expression or stimulation (Figure 1—source data 1). In cells expressing the 5-HT2C receptor, Thr383 phosphorylation showed a marked elevation after a 5-min 5-HT stimulation, which persisted 30 min after the onset of the treatment. Thr383 phosphorylation slightly but not significantly increased after the 30-min 5-HT challenge in cells expressing 5-HT4 receptor and was much less pronounced than that measured in presence of 5-HT2C receptor (Figure 1B and Figure 1—source data 1). We next examined whether 5-HT2C receptor stimulation likewise induces phosphorylation of endogenous β-arrestin2 at Thr383, but did not detect any ion signal corresponding to the phosphorylated peptide comprising this residue in cells not transfected with the YFP-β-arrestin2 plasmid. We thus generated a polyclonal antibody that specifically recognizes β-arrestin2 phosphorylated at Thr383 (see Figure 2C and Figure 2—source data 1). Western blot experiments using this antibody showed that 5-HT2C receptor stimulation induces a substantial increase in endogenous β-arrestin2 phosphorylation at Thr383, while activation of the 5-HT4 receptor did not affect its phosphorylation level (Figure 1C and Figure 1—source data 2), corroborating the MS/MS analysis of ectopic β-arrestin2 phosphorylation. Figure 1 with 5 supplements see all Download asset Open asset 5-HT2C and 5-HT4 receptor stimulation promotes β-arrestin2 phosphorylation in HEK-293 cells. (A) Ribbon diagram of rat β-arrestin2 showing the position of phosphorylated residues identified by LS-MS/MS. (B) Representative extracted ion chromatograms of the EIDIPVDTNLIEFDTNYAp383TDDDIVFEDFAR peptide from YFP-tagged β-arrestin2 in cells expressing 5-HT2C or 5-HT4 receptor and challenged with vehicle (Basal) or 5-HT (1 and 10 µM, respectively) for 5 or 30 min. Two other independent experiments performed on different sets of cultured cells yielded similar results. The histogram represents the means ± SEM of ion signal intensities of the peptide obtained in the three experiments. (C) 5-HT2C or 5-HT4 receptor expressing cells were treated as in (B). Erk1,2 activation and Thr383 phosphorylation were assessed by Western blotting using the anti-phospho-Thr202/Tyr204-Erk1/2 and the anti-phospho-Thr383 β-arrestin2 antibody, respectively. The histogram shows the means ± SEM of the anti-phospho-Thr383 β-arrestin2 immunoreactive signals (expressed in arbitrary unit) obtained in three independent experiments performed on different sets of cultured cells. One-way ANOVA: (B) F(5,12)=7.544, p=0.0020; (C) F(4,10) = 4.417, p=0.0259. *p<0.05 vs. corresponding vehicle. https://doi.org/10.7554/eLife.23777.002 Figure 1—source data 1 List of phosphorylated peptides identified from purified β-arrestin1 and β-arrestin2 by nano-LC-MS/MS. Quantitative data were used to build histogram in Figure 1B. Phosphorylated peptides were analyzed by nano-LC-MS/MS using multistage activation on the neutral loss of phosphoric acid. For each peptide, the position of modified residue(s) in the protein sequence, experimental and theoretical masses, mass deviation, charge, Mascot score and corresponding p-value are indicated. The phosphorylation index (phosphorylated peptide MS signal intensity/phosphorylated peptide MS signal intensity + non-phosphorylated peptide MS signal intensity) in cells expressing or not 5-HT2C or 5-HT4 receptor and treated with vehicle or 5-HT was also calculated for each phosphorylated peptide identified. ND: not detected. Results of one-way ANOVA for the EIDIPVDTNLIEFDTNYApTDDDIVFEDFAR peptide: F(5,12) = 7.544, p=0.020. *p<0.05 vs. vehicle. https://doi.org/10.7554/eLife.23777.003 Download elife-23777-fig1-data1-v2.xlsx Figure 1—source data 2 This file contains raw values used to build Figure 1C. https://doi.org/10.7554/eLife.23777.004 Download elife-23777-fig1-data2-v2.xlsx Figure 2 with 3 supplements see all Download asset Open asset Role of MEK in the phosphorylation of β-arrestin2 at Thr383 elicited by 5-HT2C receptor stimulation. (A) Mechanistic model of assembly of the 5-HT2C receptor/β-arrestin2/Erk module. Color code: receptor in orange, MEK in green, β-arrestin2 core in pale cyan and C-tail in cyan (the regions 351–384 and 394–419, which are not visible in 3D structure are represented by dashed lines, the region 385–393 is represented by spheres), Erk in dark red, Raf-1 RBD domain in pink. In this model, we hypothesize that Thr383 phosphorylation by MEK takes place within the assembled receptor/β-arrestin/Raf/MEK complex and results in a movement of β-arrestin2 unfolded 350–393 segment away from the first β-strand of β-arrestin, leaving space for further interaction with the receptor C-terminal domain (orange spheres) and recruitment of Erk, and its subsequent phosphorylation by MEK. For the clarity of the figure, the extremity of the β-arrestin C-tail is represented by spheres even in its unfolded state, although the real 3D structure is unknown. (B) Representative extracted ion chromatograms of the peptide in cells expressing 5-HT2C receptor, pretreated with either vehicle (control) or FR180204 (10 µM for 18 hr) or U0126 (5 µM for 30 min) or coexpressing MEK1 dominant-negative mutant (MEK1DN), and challenged with vehicle (Basal) or 5-HT (1 µM) for 30 min. The histogram represents the means ± SEM of the corresponding ion signal intensities (normalized to values in 5-HT-stimulated cells in Control condition) obtained in three independent experiments. One-way ANOVA: F(8,18) = 15.69, p<0.0001. ***p<0.001 vs. corresponding basal value. (C) YFP-tagged β-arrestin2 (wild-type or Thr383Ala mutant) purified from transfected HEK-293 cells was incubated with active MEK1 for 15 min at 37°C. When indicated, U0126 (5 µM) was included in the incubation medium. Thr383 phosphorylation was assessed by sequential immunoblotting with the antibody raised against phospho-Thr383 β-arrestin2 and the anti-β-arrestin2 antibody. Means ± SEM of results from four independent experiments are shown on the histogram. n.d.: not detectable. One-way ANOVA: F(2,9) = 352.2, p<0.0001. ***p<0.001 vs. immunoreactive signal in absence of MEK; §§§ p<0.001 vs. corresponding condition in absence of U0126. https://doi.org/10.7554/eLife.23777.010 Figure 2—source data 1 This file contains raw values used to build Figure 2B, C. https://doi.org/10.7554/eLife.23777.011 Download elife-23777-fig2-data1-v2.xlsx Role of MEK in β-arrestin2 phosphorylation at Thr383 elicited by 5-HT2C receptor stimulation Given the strong enhancement of Thr383 phosphorylation induced by the stimulation of 5-HT2C receptor that parallels the β-arrestin2-dependent receptor-operated activation of Erk1/2 (Figure 1 and Figure 1—figure supplement 1), we paid particular attention to the phosphorylation of this residue, which is located in an unfolded region of the carboxy-terminal region of β-arrestin2 easily accessible to protein kinases (Figure 1A). In an effort to identify kinase(s) involved in its phosphorylation, we first used Group-based Prediction System (GPS, v2.1), an algorithm for kinase consensus search that classifies protein kinases into a hierarchical structure with four levels and trains against the PhosphoELM database, in order to determine individual false discovery rates for each of them (Xue et al., 2008). GPS search revealed that Thr383 is a strong consensus for phosphorylation by casein kinase 2 (CK2 GPS score 5.3), consistent with previous findings (Lin et al., 2002; Kim et al., 2002). However, neither basal nor 5-HT2C receptor-elicited Thr383 phosphorylation was affected by treating cells with the selective cell-permeable CK2 pharmacological inhibitor tetrabromocinnamic acid (TBCA, Figure 2—figure supplement 1 and Figure 2—figure supplement 1—source data 1) (Pagano et al., 2007), indicating a marginal contribution of CK2 in the phosphorylation of this residue in living HEK-293 cells. GPS search also suggested that Thr383 is a potential site for phosphorylation by MAP2K1 (MEK1, GPS score 6.0). Numerous studies have shown that β-arrestins associated with a GPCR can interact with different signaling proteins including c-Src and several proteins of the Erk signaling module (Raf/MEK/Erk), and that these interactions depend on β-arrestin conformational state (Xiao et al., 2004; Gurevich and Gurevich, 2003). In line with these findings, we have recently modeled the complex between a GPCR, β-arrestin2, c-Src and the Erk module from the 3D structure of each partner, using the Protein-Protein cOmplexes 3D structure pRediction (PRIOR) docking algorithm, and validated experimentally the previously unknown interaction regions (Bourquard et al., 2015). This model predicts that the unfolded C-terminal part of β-arrestin comprising Thr383 is located in the vicinity of the MEK active site (Figure 2A), indicating a possible role of MEK bound to β-arrestin2 in its phosphorylation. Based on this model, we hypothesized that the mechanism of β-arrestin-dependent activation of Erk consists in the following steps: (i) Raf and MEK assemble to β-arrestin2 bound to agonist-stimulated receptor, resulting in MEK activation, (ii) MEK phosphorylates β-arrestin2 at Thr383, resulting in a large conformational change of the β-arrestin2 region comprising residues 350–393, (iii) the receptor C-terminus associates with β-arrestin2 using the region previously occupied by the 383–393 β-strand within the β-arrestin2 C-terminal tail and (iv) Erk binds to the complex and can be activated by MEK (Figure 2A). Consistent with this hypothesis, pretreatment of cells with the MEK pharmacological inhibitor U0126 (5 µM) abolished Thr383 phosphorylation elicited by 5-HT2C receptor stimulation (Figure 2B and Figure 2—source data 1). Expression of a MEK1 dominant-negative inhibitor (MEK1DN) also strongly reduced Thr383 phosphorylation state (Figure 2B, see also Figure 2—figure supplement 2 for controls showing strong MEK inhibition induced by both approaches, as assessed by monitoring phosphorylation of Erk1/2). In contrast, pretreating cells with FR180204 (10 µM), a selective pharmacological inhibitor of Erk1/2, did not affect Thr383 phosphorylation (Figure 2B). The ability of FR180204 treatment to inhibit Erk1/2 activity without affecting MEK activity in our experimental conditions was assessed by monitoring phosphorylation of Erk1/2 and their substrate Elk1 (Figure 2—figure supplement 2). Collectively, these results identify MEK as the major kinase involved in phosphorylation of β-arrestin2 at Thr383 upon 5-HT2C receptor stimulation. To further confirm the ability of MEK to directly phosphorylate this residue, we performed an in vitro kinase assay using purified active MEK1 and YFP-β-arrestin2 purified from transfected HEK-293 cells as substrate, followed by Western blotting using the anti-phospho-Thr383β-arrestin2 antibody. MEK1 induced a strong elevation in the immunoreactive signal that was abolished by adding U0126 in the incubation medium (Figure 2C). As expected, no increase in immunoreactive signal was observed in experiments using YFP-Thr383Ala β-arrestin2 mutant as substrate, thus validating the specificity of the antibody for β-arrestin2 phosphorylated at Thr383. This result was confirmed using a radioactive kinase assay, which also showed comparable efficacy of MEK1 to promote phosphorylation of purified β-arrestin2 and its canonical substrate Erk2 in vitro (Figure 2—figure supplement 3 and Figure 2—figure supplement 3—source data 1). β-arrestin2 phosphorylation at Thr383 neither affects translocation of β-arrestin2 to 5-HT2C and 5-HT4 receptors nor agonist-induced receptor internalization To explore whether Thr383 phosphorylation affects the recruitment of β-arrestin2 by 5-HT2C and 5-HT4 receptors, we monitored interaction between each receptor and either wild type or Thr383Ala or Thr383Asp β-arrestin2 mutants using a BRET-based assay. Exposure of cells to 5-HT induced a strong increase in the BRET signal between 5-HT2C-YFP or 5-HT4-YFP receptor and Rluc-β-arrestin2 (Figure 3A and B and Figure 3—source data 1). The 5-HT-elicited BRET signal was similar in cells expressing wild type β-arrestin2, Thr383Ala or Thr383Asp β-arrestin2 mutants, indicating that Thr383 phosphorylation does not affect β-arrestin2 translocation to either receptor. Likewise, 5-HT treatment decreased cell surface expression of 5-HT2C and 5-HT4 receptors to a similar extent in cells expressing wild type β-arrestin2, Thr383Ala or Thr383Asp β-arrestin2 mutants (Figure 3C and D), indicating that Thr383 phosphorylation does not affect agonist-induced internalization of these receptors. Figure 3 Download asset Open asset Thr383 phosphorylation underlies β-arrestin2 conformational rearrangement elicited by 5-HT2C receptor stimulation. (A, B) Translocation of wild type (WT), T383A and T383D Rluc-β-arrestin2 to Myc-5-HT2C-YFP (A) or Myc-5-HT4-YFP (B) receptors in cells treated with either vehicle (Basal) or 1 or 10 µM 5-HT, respectively, was measured by BRET. Data represent the mean ± SEM of values obtained in three independent experiments and were normalized to the BRET signals measured in 5-HT-stimulated cells expressing WT Rluc β-arrestin2. (C, D) Cell surface expression of receptors was measured in the same experimental condition by ELISA using anti-Myc antibody. Data are the mean ± SEM of values obtained in three independent experiments. They were normalized to total receptor expression level and are expressed in % of basal receptor level at the cell surface in cells expressing WT β-arrestin2. (E, F) Conformational arrangement of WT, T383A and T383D double brilliance Rluc8-β-arrestin2-RGFP elicited by 5-HT2C and 5-HT4 receptor stimulation by 5-HT (1 and 10 µM, respectively). Equivalent expression of each BRET sensor was verified by ELISA. Data represent the mean ± SEM of values obtained in three independent experiments and were normalized to the basal intra-molecular BRET signal in cells expressing WT Rluc8-β-arrestin2-RGFP. One-way ANOVA: A, F(5,12)=10.75, p=0.0004; B, F(5,12)=320.9, p<0.001; C, F(6,14)=10.82, p<0.0001; D, F(6,14)=48.52, p<0.0001; E, F(5,12)=5.136, p=0.0095; F, F(5,12)=6.436, p=0.004. *p<0.05, **p<0.01 ***p<0.001 vs. corresponding basal. https://doi.org/10.7554/eLife.23777.017 Figure 3—source data 1 This file contains raw values used to build Figure 3. https://doi.org/10.7554/eLife.23777.018 Download elife-23777-fig3-data1-v2.xlsx Impact of Thr383 phosphorylation upon β-arrestin2 conformational changes elicited by 5-HT2C and 5-HT4 receptor stimulation β-arrestins are known to undergo important conformational rearrangements upon translocation to agonist-stimulated GPCRs that are essential for their downstream action (Xiao et al., 2004; Gurevich and Gurevich, 2014). To explore the impact of Thr383 phosphorylation on conformational changes of β-arrestin2 induced by 5-HT2C receptor activation vs. 5-HT4 receptor activation, we used an optimized intramolecular BRET biosensor consisting of β-arrestin2 sandwiched between the Renilla green fluorescent protein (RGFP) and the Renilla luciferase variant Rluc8 (Kamal et al., 2009; Charest et al., 2005). When β-arrestin2 conformation changes, the distance between Rluc8 and RGFP increases or decreases leading to a BRET signal decrease or increase. Treating cells coexpressing 5-HT2C receptor and WT RLuc8-β-arrestin2-RGFP with 5-HT increased the BRET signal. No BRET increase was detected upon agonist exposure in cells coexpressing the receptor and T383A RLuc8-β-arrestin2-RGFP (Figure 3E). Mutation of Thr383 into aspartate in RLuc8-β-arrestin2-RGFP also resulted in an increase in BRET signal to a level similar to the one measured in cells expressing the wild type probe and exposed to 5-HT, and 5-HT treatment did not further enhance this elevated BRET signal (Figure 3E). In contrast, substitution of Thr383 by alanine or aspartate did not affect the increase in RLuc8-β-arrestin2-RGFP BRET signal induced by 5-HT treatment in cells expressing 5-HT4 receptors (Figure 3F). Collectively, these results indicate that β-arrestin2 conformational change elicited by 5-HT2C receptor stimulation, but not 5-HT4 receptor stimulation, depends on Thr383 phosphorylation. β-arrestin2 phosphorylation at Thr383 is essential for Erk1/2 translocation to the 5-HT2C receptor/β-arrestin2 complex As previously hypothesized, it can be envisioned that the unfolded C-terminal region of β-arrestin2 occupies the docking site of Erk1/2 located in the vicinity of MEK catalytic site in the receptor/β-arrestin2 complex (Figure 2A). Consequently, Thr383 phosphorylation status might affect Erk1/2 recruitment. In fact, our model predicts that Thr383 phosphorylation should induce a movement of the β-arrestin2 C-terminal region away from the complex leaving space for further recruitment of Erk (Figure 2A). To explore this possibility, we compared the ability of wild type and Thr383Ala and Thr383Asp β-arrestin2 to recruit Erk1/2 upon 5-HT2C receptor stimulation by co-immunoprecipitation. Treating 5-HT2C receptor-expressing cells with 5-HT increased the amount of Erk1/2 co-immunoprecipitated with β-arrestin2, an effect abolished by mutating Thr383 into Ala (Figure 4A and Figure 4—source data 1). In contrast, mutation of Thr383 into aspartate increased Erk1/2 recruitment by β-arrestin2 even in the absence of the agonist and receptor stimulation did not induce any additional effect (Figure 4A). Further supporting a role of Thr383 phosphorylation by MEK, treatment of cells with U0126 prevented recruitment of Erk1,2 by β-arrestin2 induced by 5-HT2C receptor stimulation (Figure 4—figure supplement 1 and Figure 4—figure supplement 1—source data 1). 5-HT did not promote Erk1/2 recruitment by β-arrestin2 in cells coexpressing 5-HT4 receptor and either wild type or mutated forms of β-arrestin2 (Figure 4B). Collectively, these results suggest that Thr383 phosphorylation by MEK promotes a conformational change of β-arrestin2 that facilitates Erk recruitment by the 5-HT2C receptor/β-arrestin2 complex, whereas it has no influence upon its translocation to the 5-HT4 receptor/β-arrestin2 complex. Figure 4 with 1 supplement see all Download asset Open asset Phosphorylation of β-arrestin2 at Thr383 is a necessary step in Erk1/2 recruitment to β-arrestin2 and engagement of Erk signaling by 5-HT2C receptor. (A, B) Recruitment of Erk1/2 to WT, T383A and T383D YFP-β-arrestin2 in cells expressing 5-HT2C or 5-HT4 receptor and exposed or not to 5-HT (1 and 10 µM, respectively) was assessed by co-immunoprecipitation. Immunoblots representative of three independent experiments are illustrated. The histograms represent the means ± SEM of Erk1/2 immunoreactive signals in immunoprecipitates, assessed by densitometric analysis, obtained in the three experiments. They were normalized to the amount of YFP-β-arrestin2 immunoprecipitates and expressed in % of basal level measured in cells expressing WT β-arrestin2. *p<0.05 vs. basal value in cells expressing WT β-arrestin2. (C, D) Erk1/2 activation in cells co-expressing 5-HT2C or 5-HT4 receptor and either WT, or T383A and or T383D YFP-β-arrestin2 and exposed or not to 5-HT (1 and 10 µM, respectively) for 5 or 30 min was assessed by sequential immunoblotting with the antibody recognizing phospho-Thr202/Tyr204-Erk1/2 and total Erk1/2. Immunoblots representative of three independent experiments are illustrated. The histograms represent the means ± SEM of values (normalized to the level of phosphorylated Erk1/2 in cells expressing WT β-arrestin2 and exposed to 5-HT for 5 min) obtained in the three experiments. One-way ANOVA: A, F(5,12)=4.305, p=0.00178; B, F(5,12)=0.977, p=0.47; C, F(8,18)=11.78, p<0001; D, F(8,18)=4.998, p=0.0022. *p<0.05, **p<0.01 vs. corresponding value in cells expressing WT β-arrestin2. https://doi.org/10.7554/eLife.23777.019 Figure 4—so
Arrestin
Internalization
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Arrestin
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Functional selectivity
Rhodopsin-like receptors
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Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with β-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in β-arrestin recruitment and GPCR-β-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for β-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of β-arrestin and stabilize GPCR-β-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-β-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for β-arrestin recruitment, this provides a mechanism for β-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.
Arrestin
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