Research ArticleGPCR SIGNALING

Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid μ-opioid receptor desensitization

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Science Signaling  17 Jul 2018:
Vol. 11, Issue 539, eaas9609
DOI: 10.1126/scisignal.aas9609

Controlling opioid receptor desensitization

Chronic opioid use leads to tolerance and the need for increasing doses to achieve pain relief, euphoria, and other effects. At the molecular level, tolerance occurs because of μ-opioid receptor desensitization: Opioid activation of its receptors triggers their phosphorylation and internalization, ultimately reducing the cellular response to opioids. Miess et al. investigated why high-efficacy opioids induce greater receptor internalization than lower-efficacy opioids such as morphine. The kinase GRK2 and the scaffolding protein β-arrestin regulate the desensitization of opioid receptors and other G protein–coupled receptors, and the authors assessed the recruitment of GRK2 and β-arrestin to phosphorylation site receptor mutants. GRK2 was more rapidly recruited to μ-opioid receptors by high-efficacy opioids than by morphine, and GRK2 promoted receptor desensitization to high-efficacy opioids through phosphorylation-dependent and phosphorylation-independent means. These results provide molecular insight into μ-opioid receptor desensitization, which may aid in the development of synthetic opioids that do not induce tolerance and thus have reduced potential for addiction.

Abstract

G protein receptor kinases (GRKs) and β-arrestins are key regulators of μ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser375 and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and β-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was β-arrestin to a DAMGO-activated MOR. β-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the 370TREHPSTANT379 motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and β-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.

INTRODUCTION

Opioids such as morphine are still the mainstay analgesics for the treatment of severe pain. However, the development of tolerance, addiction, and respiratory depression severely limits their utility (1). These unwanted effects of opioids are key contributors to opioid-induced overdose deaths, which have drastically increased in the last decade (2).

The μ-opioid receptor (MOR) is the G protein–coupled receptor (GPCR) targeted by morphine and most opioid analgesics (3). Since the initial observation that morphine elicited less tolerance and decreased side effects in mice lacking β-arrestin2 (4, 5), substantial efforts have focused on the development of opioid ligands that preferentially activate G protein–mediated signals over β-arrestin recruitment. These efforts have resulted in the discovery of several molecules with an improved side effect profile and increased therapeutic window (68). However, the cellular mechanisms whereby opioids mediate analgesia compared to other side effects are still not clear. MOR desensitization is considered to be the initial step for the development of tolerance. Such desensitization entails phosphorylation of the receptor, recruitment of regulatory proteins such as β-arrestins, and receptor internalization (1). Together, these regulatory processes result in a reduction of opioid response or sensitivity.

The ligand-dependent and hierarchical nature of MOR phosphorylation and its role in MOR desensitization and internalization have been previously established. Quantitative mass spectrometry and phosphorylation site-specific antibodies have identified two clusters of MOR residues—354TSST357 and 370TREHPSTANT379 within its C-terminal region—that undergo opioid-induced phosphorylation (911). Agonist-mediated phosphorylation of MOR is initiated at Ser375, but it is the ability of such agonists to induce higher-order phosphorylation on flanking residues that dictates their propensity to internalize MOR. Different opioids produce different phosphorylation patterns; multisite phosphorylation in the C-terminal region of MOR occurs robustly for agonists that induce internalization with less phosphorylation of fewer sites for those that do not (911).

Rapid desensitization of MOR coupling to membrane effectors such as voltage-gated calcium channels (VGCCs) and inwardly rectifying K channels (GIRKs; Kir3.X) also precedes internalization, but its relationship to phosphorylation events and β-arrestin recruitment remains unclear (1, 12). We have previously shown that C-terminal phosphorylation of MOR is necessary for some forms of desensitization, an effect that is also ligand-dependent (13). Mutation of all Ser and Thr (S/T) residues within the C-terminal tail of MOR completely abolishes desensitization induced by [Met5]-enkephalin (ME), but not by morphine. Introduction of an S375A mutation in transgenic mice diminishes the development of tolerance to high-efficacy opioid agonists such as [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) or etonitazene without affecting tolerance to chronic morphine (14). Compound 101 (Cmpd101), a small-molecule inhibitor that prevents G protein receptor kinase (GRK) 2/3 activation (15), only partially blocked ME-, DAMGO-, and morphine-induced MOR desensitization of GIRKs in rat and mouse locus coeruleus neurons (16), suggesting GRK2/3-independent mechanisms of MOR desensitization.

Multisite phosphorylation of MOR induced by high–intrinsic efficacy agonists such as DAMGO or ME requires GRK2/3 (11), and activation of MOR by these ligands results in β-arrestin recruitment to the receptor (1719). Overexpression of GRK2 also facilitates morphine-induced β-arrestin recruitment and MOR internalization (20), supporting a key role of this kinase in MOR regulation. However, it is still unknown what dictates GRK-MOR interactions. Moreover, Raveh et al. (21) have proposed that GRKs may act to desensitize GIRK channels through a mechanism that is independent of their kinase activity through competition for the βγ subunits of the G protein that activate these channels. Thus, GRKs and arrestins are critical regulatory proteins for which interaction with MOR before internalization has major implications for opioid signaling. However, the molecular determinants that control these interactions are still elusive. How phosphorylation regulates these interactions and what relevance this has for MOR desensitization are questions that remain to be addressed.

In the present study, we systematically assessed how specific multisite phosphorylation controlled the dynamics of GRK and β-arrestin interactions with MOR and showed how such phosphorylation mediates receptor desensitization. Using bioluminescence and Förster resonance energy transfer (BRET and FRET) and β-galactosidase (β-Gal) complementation technology, we showed that the kinetics of GRK2/3 recruitment to a DAMGO-activated MOR were faster than the kinetics of β-arrestin recruitment. β-Arrestin recruitment required GRK2 activity and MOR phosphorylation, whereas GRK recruitment also depended on the integrity of the phosphorylation sites in the C terminus of MOR. Although the 370TREHPSTANT379 motif was required for effective recruitment of GRKs and β-arrestins, the 354TSST357 region participated in the long-term stability of such interactions. Both GRKs and β-arrestins showed residual recruitment to a receptor with mutations at all C-terminal phosphosites. Using complementary patch-clamp approaches, we unraveled a fast desensitization event that was independent of MOR phosphorylation and a sustained desensitization component, which required GRK2-mediated phosphorylation of the 370TREHPSTANT379 motif and a potential soluble factor. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.

RESULTS

Agonist-dependent recruitment of β-arrestins to wild-type MOR

We have developed complementary approaches that allow for the systematic investigation of the dynamics and mechanisms of β-arrestin recruitment to MOR. FRET and BRET allow the real-time assessment of the recruitment of a fluorescently tagged β-arrestin [cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), respectively] to a C-terminally tagged MOR [YFP or Renilla luciferase 8 (RLuc8), respectively]. In addition, we have also developed a β-Gal complementation approach to measure steady-state interactions between β-arrestin and MOR. Thus, whereas FRET and BRET provide information about the dynamics of β-arrestin recruitment at early time points, the β-Gal complementation assay provides further insight into the stability of these interactions (Fig. 1A).

Fig. 1 Agonist-dependent recruitment of β-arrestin1/2 to WT MOR.

(A) Schematic representation of the approaches used in this study: BRET, FRET, and β-Gal complementation. (B) BRET measurements in human embryonic kidney (HEK) 293 cells expressing MOR-RLuc8 and β-arrestin2–YFP and stimulated with 1 μM DAMGO or 1 μM morphine for 20 min before addition of 30 μM naloxone (n = 4 independent experiments). (C) FRET analysis of HEK293 cells expressing MOR-YFP and β-arrestin2–CFP and stimulated with 10 μM DAMGO or 30 μM morphine for 1 min before agonist washout (n = 6 independent experiments). (D and E) BRET analysis of HEK293 cells expressing MOR-RLuc8 and β-arrestin1/2–YFP and stimulated for 10 min with increasing concentrations of DAMGO (D) or morphine (E) (n = 6 independent experiments). (F and G) β-Gal complementation analysis of HEK293 cells expressing MOR–β-Gal1–44 and β-arrestin1/2–β-Gal45–1043 and stimulated with increasing concentrations of DAMGO (F) or morphine (G) for 1 hour (n = 4 to 6 independent experiments). Raw BRET/FRET ratio of vehicle-treated cells was subtracted, and raw bioluminescence data from β-Gal were normalized to vehicle-treated cells. Data points represent mean ± SEM of the indicated number of experiments.

The high–intrinsic efficacy agonist DAMGO and the low–intrinsic efficacy opiate morphine (both at 1 μM) increased the BRET signal between MOR-RLuc8 and β-arrestin2–YFP, which peaked within 1 min, although the signal obtained with morphine was smaller than that obtained with DAMGO (Fig. 1B). This signal was blocked by the addition of the antagonist naloxone (30 μM), indicating the specificity and reversibility of the response (Fig. 1B). FRET measurements between MOR-YFP and β-arrestin2–CFP showed similar results (Fig. 1C) (22). Activation of MOR with saturating concentrations of DAMGO (10 μM) promoted β-arrestin2 recruitment to the receptor that peaked within 1 min and was reversed after agonist removal. Saturating concentrations of morphine (30 μM) also induced β-arrestin2–CFP recruitment to MOR-YFP, although with a lower FRET signal (Fig. 1C).

We then constructed concentration-response curves for β-arrestin1/2 recruitment using BRET (10-min incubation) or β-Gal complementation assays (1-hour incubation) (Fig. 1, D to G). DAMGO induced a concentration-dependent recruitment of β-arrestin1/2 in both assays with similar potencies (Table 1). In contrast, morphine did not significantly induce β-arrestin1 recruitment and caused only a partial recruitment of β-arrestin2 with similar potency to DAMGO (Table 1). These results are in agreement with previous findings that show a compromised ability of morphine to recruit β-arrestins in the absence of GRK overexpression (18, 20).

Table 1 Potency (pEC50) and maximal response (Emax) of β-arrestin1/2 recruitment to WT and phosphorylation-deficient MOR mutants.

NA, not available.

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Recruitment of β-arrestins to phosphorylation-deficient MOR mutants

To map putative phosphate acceptor sites controlling MOR phosphorylation, we generated phosphorylation-deficient MOR mutants and directly assessed the correlation between phosphorylation and recruitment of regulatory proteins. Receptor phosphorylation at candidate sites was evaluated using phosphosite-specific antibodies (Fig. 2A). Mutations of S/T residues to alanine (A) in different regions of wild-type (WT) MOR generated the following phosphorylation-deficient MOR mutants: TSST-4A (mutations in the 354TSST357 cluster), S375A (Ser375 mutation), STANT-3A (mutations in the 375STANT379 cluster), TREHPSTANT-4A (mutations in the 370TREHPSTANT379 motif), and 11S/T-A (mutation of all 11 potential C-terminal S/T phosphorylation sites) (Fig. 2B).

Fig. 2 C-tail phosphorylation of MOR.

(A) Schematic representation of the C-tail domain of mouse MOR with potential phosphate acceptor sites depicted in gray and phosphosite-specific antibodies against pSer/pThr residues depicted in black (pSer356/pThr357, pThr370, pSer375, pThr376, and pThr379). The epitope recognized by the phospho-independent antibody UMB-3 is underlined. (B) C-tail sequences of WT MOR and phosphorylation-deficient MOR mutants are also illustrated. S/T residues depicted in black were mutated to A depicted in gray as shown in each of the mutants. (C) Characterization of phosphosite-specific antibodies using Western blot analysis. HEK293 cells stably expressing hemagglutinin (HA)–tagged MOR WT, MOR TSST-4A, MOR S375A, MOR TREHPSTANT-4A, or MOR 11S/T-A were stimulated with 10 μM DAMGO or morphine for 30 min at 37°C. Cells were lysed and immunoblotted with anti-pSer356/pThr357, anti-pThr370, anti-pSer375, anti-pThr376, or anti-pThr379 antibodies. Blots were stripped and reprobed with the phosphorylation-independent MOR antibody UMB-3 or with HA antibody to confirm equal loading (n = 3 independent experiments).

Consistent with earlier observations (10, 11), no constitutive phosphorylation of Ser356/Thr357, Thr370, Ser375, Thr376, or Thr379 was observed. Agonist stimulation with morphine resulted in phosphorylation of Ser375 and induced only weak phosphorylation of Thr370, Thr376, and Thr379. In contrast, DAMGO resulted in robust MOR phosphorylation at all sites in the 370TREHPSTANT379 domain (Fig. 2C) (10, 11). Equivalent receptor loading was confirmed by detecting a distinct nonphosphorylated epitope in the cytoplasmic tail.

Expanding from previous studies, we generated a phosphosite-specific antiserum against Ser356 and Thr357 of the 354TSST357 motif of the MOR C-terminal tail (pSer356/pThr357). DAMGO stimulation induced very weak phosphorylation of these residues, which was abolished in the TSST-4A mutant. Morphine did not promote phosphorylation of any of these residues (Fig. 2C). In addition, mutation of the 354TSST357 motif in the TSST-4A mutant produced a modest decrease in the phosphorylation at other S/T sites by both ligands. These data exclude both Ser356/Thr357 as prominent initial sites for agonist-induced MOR phosphorylation but highlight the idea that the integrity of the 354TSST357 cluster might be important for regulatory events that depend on robust phosphorylation within the 370TREHPSTANT379 domain.

Point mutation of Ser375 (S375A mutant) within the 375STANT379 motif prevented detectable phosphorylation at Ser356/Thr357, Thr376, and Thr379, and also substantially reduced phosphorylation at Thr370 (Fig. 2C). This confirms Ser375 as the initial residue for agonist-specific hierarchical MOR phosphorylation as previously reported (10, 11). As expected, detection of phosphorylated MOR by any of the phospho-specific antibodies was blocked by mutation of the 370TREHPSTANT379 motif or mutation of all S/T residues in the cytoplasmic tail (11S/T-A mutant) (Fig. 2C).

To directly assess the relationship between receptor phosphorylation and the recruitment of β-arrestin1/2, we generated phosphorylation-deficient MOR mutants (Fig. 2A) suitable for BRET, FRET, and β-Gal complementation assay. Correct expression and function of these mutants were evaluated by anti-FLAG enzyme-linked immunosorbent assay (ELISA) and inhibition of forskolin-induced cyclic adenosine monophosphate (cAMP) of FLAG-tagged MOR constructs without C-terminal tags (fig. S1, A and B, and table S1).

Mutation of the 354TSST357 cluster (TSST-4A) did not affect DAMGO-induced β-arrestin1/2 recruitment as measured using BRET (Fig. 3, A to C, and Table 1). However, β-Gal complementation showed that deletion of this cluster decreased the absolute recruitment of β-arrestin1/2 (Emax) without altering the potency of DAMGO (Fig. 3, B and C, and Table 1). β-Gal complementation requires the two fragments to be in the correct relative orientation to form a functional enzyme, which has been suggested to occur upon “stable” interactions (30 to 90 min). In contrast, for FRET and BRET to occur, the relative orientation between the receptor and β-arrestin is under fewer constraints. Thus, these data suggest that although the initial recruitment of β-arrestins to the receptor may not be affected by mutation of the 354TSST357 motif, the stability of this interaction is affected upon mutation of this motif to alanine. Thus, phosphorylation of the 354TSST357 region, although not directly involved in β-arrestin recruitment, may participate in the stability of the β-arrestin/MOR complex.

Fig. 3 Recruitment of β-arrestin1/2 to phosphorylation-deficient MOR mutants.

(A) BRET analysis of HEK293 cells transiently transfected with MOR-RLuc8 WT or phosphorylation-deficient mutants and β-arrestin1/2–YFP and stimulated for 30 min with 1 μM DAMGO (n = 3 to 5 independent experiments). Raw BRET ratio of vehicle-treated cells was subtracted, and data represent mean ± SEM. Area under the curve (AUC) of BRET signal is shown as a percentage of the maximal response to DAMGO in the WT receptor and represents means ± SEM. All responses are significant against vehicle; ^ denotes significance compared to WT (P < 0.01) by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test; * denotes significance of DAMGO compared to morphine (P < 0.01) by two-way ANOVA with Sidak’s multiple comparison test. (B) BRET analysis of HEK293 cells expressing MOR-RLuc8 WT or phosphorylation-deficient mutants and β-arrestin2–YFP or β-Gal complementation analysis of cells expressing MOR–β-Gal1–44 WT or phosphorylation-deficient mutants and β-arrestin2–β-Gal45–1043. Cells were stimulated with increasing concentrations of DAMGO for 10 min or 1 hour, respectively. Data were normalized to vehicle-treated cells and represent mean ± SEM (n = 3 to 6 independent experiments). (C) BRET analysis of HEK293 cells expressing MOR-RLuc8 WT or phosphorylation-deficient mutants and β-arrestin1–YFP or β-Gal complementation analysis of cells expressing MOR–β-Gal1–44 WT or phosphorylation-deficient mutants and β-arrestin1–β-Gal45–1043. Cells were stimulated with increasing concentrations of DAMGO for 10 min or 1 hour, respectively. Data were normalized to vehicle-treated cells and represent mean ± SEM (n = 3 to 6 independent experiments). (D) FRET analysis of HEK293 cells expressing MOR-YFP phosphorylation-deficient mutants and β-arrestin2–CFP and stimulated with 10 μM DAMGO or 30 μM morphine for 1 min before agonist washout (left, n = 14 independent experiments for WT and n = 15 independent experiments for S375A; right, n = 17 independent experiments for WT and 11S/T-A).

Mutation of the 375STANT379 cluster (STANT-3A) or 370TREHPSTANT379 affected DAMGO-induced β-arrestin1/2 recruitment to MOR in both BRET and β-Gal complementation assays (Fig. 3, A to C), respectively. Although the potency of DAMGO was unaffected for this mutant, there was a ~50% reduction of the maximal effect of this ligand (Table 1). These results suggest that the 375STANT379 motif in conjunction with Thr370 constitutes a key region for receptor-arrestin interactions, most likely participating in the initial recruitment of β-arrestin to the activated receptor. Within this region lies Ser375, which drives the hierarchical phosphorylation of MOR, and which affected the dynamics of β-arrestin2 recruitment when mutated. Although the BRET response to DAMGO within the first 5 min after stimulation was similar to the WT receptor, the signal rapidly decayed to levels similar to those measured for the STANT-3A mutant (Fig. 3A). Furthermore, although the potency of DAMGO at this mutant was similar to that of the WT receptor, its maximal effect was reduced by 30% (Table 1). As expected, the β-Gal complementation assay revealed that DAMGO-induced recruitment to the MOR S375A mutant was reduced (Fig. 3, B and C, and Table 1). In addition, FRET analysis of MOR S375A-YFP and β-arrestin2–CFP also showed that this mutation substantially attenuated β-arrestin2 recruitment (Fig. 3D). Together, these results not only support the observations that phosphorylation of Ser375 serves as an initial residue for multisite phosphorylation but also suggest that this site is key to prolonging the interaction between MOR and β-arrestins. Finally, we assessed β-arrestin2 recruitment to a mutant receptor where all the phosphorylation sites of the C-tail have been mutated to alanine (11S/T-A). As expected, DAMGO-induced β-arrestin1/2 recruitment to this mutant was significantly compromised in all the three assays (Fig. 3, A to D, and Table 1).

We observed that although the DAMGO-induced BRET signal was reduced in the STANT-3A and 11S/T-A mutants, it was not completely abolished and it still increased from basal levels upon addition of the agonist. Addition of the antagonist naloxone (30 μM) reversed the BRET signal to basal levels (fig. S2). A similar effect was observed in the FRET assay after agonist removal (Fig. 3D), although recruitment of β-arrestin2 was not detected for the two phosphorylation-deficient mutants in the β-Gal complementation assay. These data suggest that even in the absence of phosphorylation sites in the C-tail of the MOR, β-arrestins can be transiently recruited to the receptor upon agonist stimulation.

We also investigated the effects of the above mutations on morphine-induced β-arrestin2 recruitment. As observed in the WT receptor, the response induced by morphine was significantly weaker than that induced by DAMGO (Fig. 3A and Table 1). However, the effect of all mutations on morphine’s response mirrored those observed for the high-efficacy agonist DAMGO.

Because β-arrestin recruitment precedes MOR internalization (1), we assessed the ability of DAMGO to promote endocytosis of MOR phospho-deficient mutants using BRET, cell-surface ELISA, and confocal imaging. Stimulation of the WT receptor with DAMGO, but not with morphine, induced an increase in the BRET signal between MOR-RLuc8 and the early endosome resident protein Rab5a (tagged with Venus) (Fig. 4A and fig. S2B). DAMGO-induced internalization was also observed in cell-surface ELISA assay, which quantifies agonist-induced reduction of cell-surface receptor (Fig. 4B). Confocal imaging of the HA-tagged MOR (fig. S1C) also confirmed the internalization induced by DAMGO. Mutations of the 354TSST357motif had a weak effect on DAMGO-induced MOR internalization (Fig. 4, A and B); however, the S375A, STANT-3A, TREHPSTANT-4A, or 11S/T-A mutations severely impaired receptor endocytosis. These results are consistent with the β-arrestin recruitment data and support the requirement of a strong and sustained MOR–β-arrestin interaction to drive receptor internalization.

Fig. 4 Internalization of phosphorylation-deficient MOR mutants.

(A) Internalization BRET assay in HEK293 cells transiently transfected with MOR-RLuc8 WT or phosphorylation-deficient mutants and the early endosome marker Rab5a-Venus and stimulated with 1 μM DAMGO. BRET ratio of vehicle-treated cells was subtracted. Data points represent mean ± SEM (n = 3 to 4 independent experiments). (B) HEK293 cells stably expressing the HA-tagged MOR WT, MOR TSST-4A, MOR S375A, MOR TREHPSTANT-4A, or MOR 11S/T-A were preincubated with HA antibody and stimulated with 10 μM DAMGO for 30 min. Receptor sequestration, quantified as the percentage of residual cell-surface receptors on agonist-treated cells, was measured by ELISA (n = 3 independent experiments).

Role of GRKs in β-arrestin recruitment

Agonist-induced phosphorylation of MOR is mediated by GRKs. In particular, GRK2 and GRK3 are mainly involved in MOR phosphorylation upon stimulation with high-efficacy agonists, such as DAMGO (11). We thus investigated the influence of overexpression, knockdown, and pharmacological inhibition of GRK2/3 on β-arrestin recruitment using BRET and β-Gal complementation approaches. As expected, overexpression of GRK2/3 resulted in increased efficacy and potency of DAMGO- and morphine-induced recruitment of β-arrestin1 (fig. S3A) and β-arrestin2 (Fig. 5, A to D, and Table 2). In addition, and extending a previous study showing that GRK2 overexpression increases morphine-induced phosphorylation of Ser375 (10), we showed that GRK2/3 overexpression facilitated morphine-induced multisite phosphorylation of Thr370 and Thr379 in the 370TREHPSTANT379 motif (Fig. 5E). Depletion of endogenous GRK2 or GRK3 alone by siRNA (fig. S4E) or by overexpression of a catalytically inactive GRK2 (GRK2 K220R and GRK2-DN) had a small effect in reducing the efficacy of DAMGO and morphine to recruit β-arrestin2 (Fig. 5, A and F, and Table 2). Inhibition by the GRK2/3 inhibitor Cmpd101 (15) resulted in inhibition of DAMGO-induced β-arrestin2 recruitment (Fig. 5G). In agreement with the β-arrestin recruitment data and with previous reports, MOR internalization in response to both DAMGO and morphine was enhanced by GRK2 overexpression and abolished by incubation with Cmpd101, as assessed by BRET between MOR-RLuc8 and Rab5a-Venus (fig. S3B). Together, these results illustrate the key role of GRK2/3 in the recruitment of β-arrestins and receptor endocytosis induced by opioid agonists.

Fig. 5 Role of GRKs in β-arrestin recruitment.

(A and B) BRET analysis of HEK293 cells expressing MOR-RLuc8 and β-arrestin2–YFP; cotransfected with pcDNA3 (mock), GRK2-WT, or GRK2-DN (dominant negative); and stimulated for 10 min with increasing concentrations of DAMGO (A) or morphine (B), and the BRET signal was measured after stimulation (n = 3 independent experiments). (C and D) β-Gal complementation analysis of HEK293 cells expressing MOR–β-Gal1–44 and β-arrestin1/2–β-Gal45–1043; cotransfected with pcDNA3 (mock), GRK2, or GRK3; and stimulated with increasing concentrations of DAMGO (C) or morphine (D) for 1 hour (n = 3 to 6 independent experiments). For BRET, the ratio of vehicle-treated cells was subtracted, and for β-Gal complementation, data were normalized to vehicle-treated cells. Data points represent mean ± SEM. (E) HEK293 cells expressing MOR alone (control) or in combination with pcDNA3 (mock), GRK2, GRK3, or both were stimulated with 10 μM morphine for 30 min at 37°C, lysed, and immunoblotted with anti-pThr370, anti-pSer375, or anti-pThr379 antibodies. Blots were stripped and reprobed with the phosphorylation-independent HA antibody to confirm equal loading (n = 3 independent experiments). (F) β-Gal complementation analysis of HEK293 cells expressing MOR–β-Gal1–44 and β-arrestin1/2–β-Gal45–1043; cotransfected with scrambled (SCR), GRK2, or GRK3 small interfering RNA (siRNA); and stimulated with increasing concentrations of DAMGO for 1 hour (n = 4 independent experiments). The raw BRET ratio of vehicle-treated cells was subtracted, and the raw bioluminescence data from β-Gal were normalized to vehicle-treated cells. (G) BRET analysis of HEK293 cells expressing MOR-RLuc8 and β-arrestin2–YFP and pretreated with vehicle or 30 μM Cmpd101 for 30 min before being stimulated with 1 μM DAMGO or 1 μM morphine (n = 4 independent experiments). AUC is expressed as a percentage of the maximal response of DAMGO in the control and is shown as the mean ± SEM. ^ denotes significance compared to control (P < 0.01) by two-way ANOVA with Dunnett’s multiple comparison test.

Table 2 Potency (pEC50) and maximal response (Emax) of β-arrestin2 recruitment with GRK overexpression (OE) or knockdown (KD).
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GRK recruitment to activated WT and phosphorylation-deficient MOR mutants

To understand the dynamics and mechanisms of GRK recruitment to MOR, we developed FRET, BRET, and β-Gal complementation approaches with the same donor-acceptor pairs used in the β-arrestin assays described above. Real-time measurements of FRET between MOR-YFP and GRK2-mTurquoise or BRET between MOR-RLuc8 and GRK2-Venus showed that GRK2 recruitment to the activated receptor occurs faster than β-arrestin translocation (fig. S4D) and reversed to basal upon agonist removal or addition of 30 μM naloxone (Fig. 6, A and B). Concentration-response curves constructed using BRET or β-Gal complementation assays estimated a potency of 0.6 μM for DAMGO and 0.43 μM for morphine (Fig. 6, C and D, and Table 3). Similar results were obtained for GRK3 in the β-Gal complementation assay (fig. S4, A and B). Cmpd101 prevented GRK2 recruitment to activated MOR (Fig. 6E), suggesting that an active GRK2 is required for its interaction with MOR.

Fig. 6 GRK recruitment to activated WT MOR.

(A) FRET analysis of HEK293 cells expressing MOR-YFP and GRK2-mTurquoise and stimulated with 10 μM DAMGO or 30 μM morphine for 1 min before agonist washout (n = 6 independent experiments). (B and C) BRET analysis of HEK293 cells expressing MOR-RLuc8 and GRK2-Venus and stimulated with 1 μM DAMGO or morphine for 20 min before the addition of 30 μM naloxone (B) or for 10 min with increasing concentrations of DAMGO and morphine (C) (both n = 3 independent experiments). (D) β-Gal complementation analysis of HEK293 cells expressing MOR–β-Gal1–44 and GRK2–β-Gal45–1043 and stimulated with increasing concentrations of DAMGO or morphine for 1 hour (n = 4 independent experiments). For BRET or FRET, data from vehicle-treated cells were subtracted, and for β-Gal complementation, the data were normalized to vehicle-treated cells. Data represent mean ± SEM. (E) BRET analysis of HEK293 cells expressing MOR-RLuc8 and GRK2-Venus and preincubated with control or 30 μM Cmpd101 for 30 min before stimulation with 1 μM DAMGO or morphine. The 10-min AUC was quantified and is expressed as a percentage of the maximal response of the control-treated DAMGO response (n = 4 independent experiments). Data are expressed as mean ± SEM. ^ denotes significance versus control (P < 0.01) by two-way ANOVA with Dunnett’s multiple comparison test.

Table 3 Potency (pEC50) and maximal response (Emax) of GRK2 recruitment to phosphorylation-deficient MOR mutants.
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We next sought to understand the role of phosphorylation within MOR C-tail on GRK2 translocation. We obtained similar results to the β-arrestin recruitment data described above. Namely, mutation of the 354TSST357 motif (TSST-4A) did not affect early BRET measurements (Fig. 7, A and B, and Table 3), whereas it decreased GRK2 recruitment as measured by β-Gal complementation (Fig. 7C and Table 3). These results again suggest a role of this region in the stability of interactions between the receptor and regulatory proteins. The S375A, STANT-3A, and 11S/T-A mutations all substantially reduced GRK2 recruitment in both assays (Fig. 7, A to C). Similar results were obtained for GRK3 in the β-Gal complementation assay (fig. S4C).

Fig. 7 GRK2 recruitment to phosphorylation-deficient MOR mutants.

(A and B) BRET analysis of HEK293 cells transiently expressing MOR-RLuc8 WT or phosphorylation-deficient mutants and GRK2-Venus and stimulated for 30 min with 1 μM DAMGO (A) or for 10 min with increasing concentrations of DAMGO (B) (both n = 3 independent experiments). The BRET ratio of vehicle-treated cells was subtracted, and data represent mean ± SEM. The AUC is expressed as a percentage of the maximal response to DAMGO by the WT receptor and represents mean ± SEM. All responses are significant compared to vehicle; ^ denotes significance compared to WT (P < 0.01) by two-way ANOVA with Dunnett’s multiple comparison test; * denotes significance of DAMGO compared to morphine (P < 0.01) by two-way ANOVA with Sidak’s multiple comparison test. (C) β-Gal complementation analysis of HEK293 cells expressing MOR–β-Gal1–44 WT or phosphorylation-deficient mutants and GRK2–β-Gal45–1043 and stimulated with increasing concentrations of DAMGO for 1 hour (n = 3 to 4 independent experiments). Data were normalized to vehicle-treated cells and represent the mean ± SEM. (D) FRET analysis of HEK293 cells expressing MOR-YFP phosphorylation-deficient mutants and GRK2-mTurquoise and stimulated with 10 μM DAMGO or 30 μM morphine for 1 min before agonist washout (left, n = 14 independent experiments for WT and n = 17 independent experiments for S375A; right, n = 14 independent experiments for WT and n = 13 independent experiments for 11S/T-A).

FRET experiments using the S375A and 11S/T-A mutants and GRK2-mTurquoise showed that agonist-induced recruitment of GRK2 to phosphorylation-deficient mutants was reversible after agonist washout (Fig. 7D). Although the BRET signal was reduced for the STANT-3A and 11S/T-A mutants, it was not completely abolished and it still increased from basal levels upon addition of the ligand, and only the addition of the antagonist naloxone returned the BRET signal to basal levels (fig. S4C). These data support the notion that even in the absence of phosphorylation sites at the C-tail of the MOR, GRKs and β-arrestins can be transiently recruited to the receptor upon agonist stimulation.

Desensitization of phosphorylation-deficient MOR mutants

Phosphorylation sites in the vicinity of 354TSST357 and 375STANT379 play important roles in MOR desensitization. Using conventional whole-cell mode of patch-clamp recording, Birdsong et al. (23) have shown that the STANT-3A mutation and, more critically, STANT-3A plus TSST-4A impair desensitization by ME in neurons. By contrast, using perforated patch-clamp recording to limit disruption of the cytoplasmic milieu, we have previously reported that mutation of 6 S/T residues, including STANT, abolishes ME-induced internalization but does not inhibit desensitization and that impairment of ME-induced desensitization requires mutation of all 11 S/T residues in the C-tail (13). We therefore compared desensitization of the STANT-3A and TSST-4A mutants using both whole-cell and perforated patch-clamp recording. As previously reported (13), an initial rapid component of GIRK desensitization (with a time constant of about 2 to 4 s) was observed upon application of ME (10 μM) for both TSST-4A and STANT-3A mutants that was not significantly different from WT MOR (fig. S5, A and C). This rapid component was not observed using a concentration of ME below 100 nM and was less prominent during application of morphine than ME (10 μM; fig. S5C). This effect most likely reflects GIRK regulation by GRK2, which is recruited to the receptor-channel complex by ME but less effectively by morphine, as suggested previously (21). However, it should be noted that the potency of ME to induce this rapid desensitization component could not be resolved because the on-rate of ME-activated GGIRK overlapped substantially at low concentrations with the on-rate of the declining component (fig. S5A).

Using perforated patch clamp, both STANT-3A and TSST-4A mutants showed acute desensitization upon exposure to supramaximal concentrations of both ME and morphine (10 μM) similar to WT MOR (Fig. 8, A to D). GIRK conductance acutely declined during the 5-min exposure to both ME and morphine in cells expressing the mutants or WT. As previously reported, the decline in peak GIRK conductance is an insensitive measure of receptor desensitization because of the large receptor reserve present in these cells (13, 24). Desensitization of the response to a submaximal concentration of ME (10 nM) was significant for TSST-4A, STANT-3A, and WT MOR. As previously reported (13), a smaller component of heterologous desensitization of responses to somatostatin acting on somatostatin receptors natively expressed in AtT20 cells was observed in all mutants (fig. S5E). By contrast, desensitization by ME was significantly reduced under whole-cell patch-clamp recording in the STANT-3A mutant but was maintained in TSST-4A and WT MOR (Fig. 8, B and E, and fig. S6, A to E). These results suggest that cytoplasmic desensitization mediators diffuse out of cells during whole-cell but not perforated patch-clamp recording conditions.

Fig. 8 Acute desensitization in perforated patch-clamp compared to whole-cell patch-clamp recording mode by different MOR mutants.

Extent of desensitization was determined by exposure to a submaximal concentration of ME (M; 10 nM, in red) before and after exposure to supramaximal concentrations of ME (10 μM, orange) as shown in (A) to (C). (A) Exemplar records of ME-induced desensitization of GGIRK mediated by WT and STANT-3A mutant receptors using perforated patch clamp. (B and C) Exemplar records of ME-induced desensitization of GGIRK mediated by WT or STANT-3A mutant receptors using whole-cell patch-clamp (B) or perforated patch-clamp mode after pretreatment with 30 nM calphostin C (C). (D to F) Comparison of acute desensitization using whole-cell and perforated patch-clamp recordings of different mutants (n = 5 times for each group). All scale bars represent 0.2 nS and 1 min. Two-way ANOVAs for (D) to (F) were all significant for main effects. Post hoc comparisons (Bonferroni-corrected) were significant where shown (****P < 0.0001). In (D), postdesensitized compared with predesensitized response (100%) is shown; the main effect of ME compared to morphine was not significant (n.s.; P > 0.05).

We and others have previously reported that inhibition of protein kinase C (PKC) impairs MOR desensitization (13, 25). Moreover, we have previously reported that PKCα causes restricted receptor mobility upon activation by morphine but not DAMGO (26). To assess the role of PKC, cells were preincubated with the PKC inhibitor calphostin C and exposed to the inhibitor throughout recordings. Calphostin C (30 nM) did not affect desensitization by ME in WT or TSST-4A MOR but significantly reduced ME-induced desensitization of STANT-3A (Fig. 8, C and F). These data suggest that the ME-induced desensitization by soluble mediators of the STANT-3A mutant is sensitive to PKC inhibition, in a similar manner as previously observed for morphine at the WT MOR (13).

DISCUSSION

This study identified the role of multisite phosphorylation of MOR in promoting receptor interactions with GRKs and β-arrestins and mediating rapid receptor desensitization. We systematically mutated the 11 S/T residues present in the C-tail of MOR. The use of complementary approaches to measure protein-protein interactions enabled the assessment of the dynamics of complex formation and provided key information about the roles that different C-terminal motifs play in such dynamic interactions.

The 354TSST357 cluster in the proximal region of the C-tail of MOR did not participate in receptor internalization (Fig. 4, A and B) (9). However, Birdsong et al. (23) identified the TSST region as a major mediator of the generation of the high-affinity state of the receptor upon high-efficacy agonist stimulation, suggesting that phosphorylation of this motif can have an allosteric effect on ligand binding. Our data showed that although mutation of the 354TSST357motif (TSST-4A) did not abolish phosphorylation, it did induce a detectable decrease in multisite phosphorylation induced by DAMGO (Fig. 2C). For this mutant, recruitment of GRK2/3 and β-arrestins within the first 30 min remained unaffected (Figs. 3, A to C, and 7, A and B), whereas no detectable recruitment occurred after 1-hour incubation, as observed using the β-Gal complementation approach (Figs. 3, B and C, and 7C). Moreover, desensitization of TSST-4A MOR was indistinguishable from that of the WT receptor (Fig. 8, D to F). Together, these results suggest that the TSST region, by allosterically modulating ligand binding, participates in the stability of the interaction of MOR with regulatory proteins but has no impact on MOR desensitization and internalization.

Our data support previous findings suggesting that Ser375 serves as an initial residue that drives hierarchical multisite phosphorylation of MOR (11, 27). As we have previously described, single mutation of this residue severely compromised phosphorylation of other S/T residues (Fig. 2B). Moreover, phosphorylation of Ser375 substantially affected the dynamics of recruitment of GRK2 and β-arrestin (Figs. 3A and 7A). The S375A MOR mutant recruited GRK2 and β-arrestin at early time points (5 min); however, the interaction of these proteins with the mutant receptor was transient. Using BRET, we detected a steady decrease in β-arrestin recruitment after 5 min, and the β-Gal complementation assay showed no recruitment after 1-hour incubation (Figs. 3, A to C, and 7, A to C). The use of complementary assays that rely on different stability constraints (β-Gal complementation compared to FRET and BRET) enabled the detection of these different dynamics dictated by receptor phosphorylation. The compromised internalization of S375A also suggests that robust phosphorylation and stable interactions with GRK2 and β-arrestins are required to drive MOR endocytosis. Ser375 is the first residue of the 375STANT379 motif, which has previously been reported to play a key role in receptor internalization (9), although conflicting results have been reported regarding the ability of this mutant to desensitize (13, 23). In the present study, we reconciled the differences observed with regard to the desensitization of this mutant (Fig. 8, A and B). Our data suggest that the 375STANT379 region is crucial for regulating MOR desensitization. High-efficacy agonists like DAMGO and ME regulate MOR desensitization by a GRK2/β-arrestin–mediated mechanism upon phosphorylating crucial sites within this region. Moreover, the observation that the compromised desensitization of the STANT-3A mutant is only detectable when using whole-cell patch clamp further suggests that a soluble (cytosolic) factor mediates this desensitization. In agreement with this notion, we showed that GRK2/3 and β-arrestin recruitment and receptor internalization of the STANT-3A MOR mutant were severely compromised (Figs. 3, A to C; 4, A and B; and 7, A to C). The key role of the 375STANT379 region in the regulation of MOR is also illustrated by the results obtained upon mutation of all S/T residues within the C-tail of MOR (11S/T-A) (Figs. 3, A to D; 4, A and B; and 7, A to D).

Because of its limited ability to induce multisite phosphorylation of MOR (Fig. 2B), morphine induced weak recruitment of β-arrestins (Figs. 1, B to G, and 5, B and D). Overexpression of GRK2/3 facilitated phosphorylation of Thr370 and Thr379 (in addition to Ser375) by morphine, which resulted in both increased β-arrestin recruitment and MOR internalization (Fig. 5E and fig. S3, A and B). These results highlight that the cellular effect of a particular ligand (in this case, morphine) depends on the cellular context and that differences in expression levels of signaling and regulatory proteins will influence the downstream events of receptor activation in a particular cell type. For example, different expression levels of GRK2 may explain why morphine internalizes MOR in striatal neurons (28) but not in other neurons (29), although this hypothesis remains to be confirmed.

An important finding of the current study is that even when all S/T residues of the C-tail of MOR were mutated to Ala, we detected GRK2 and β-arrestin recruitment using BRET and FRET approaches but not when using the β-Gal complementation assay. These signals were abolished by the addition of the antagonist naloxone or by agonist washout (figs. S2A and 4C). These results suggest that both GRK2 and β-arrestins can be transiently recruited to the activated MOR independently of receptor phosphorylation. Phosphorylation-independent recruitment of β-arrestins has been reported for several GPCRs (30, 31). Moreover, this recruitment of GRK2 is likely to mediate the rapid component of GIRK desensitization (21), which still occurred in the STANT-3A mutant (fig. S5C).

GRKs play a key role in GPCR regulation by phosphorylating activated receptors, uncoupling them from G proteins, and facilitating interactions with β-arrestins to promote alternative signaling and/or receptor endocytosis. Our results show that GRK2 activity was required for efficient arrestin recruitment and MOR internalization (Fig. 5, A to G, and fig. S3, A and B). An unexpected result from our studies was that the interaction between GRK2 and the activated MOR also seemed to depend on the integrity of the phosphorylation sites (Fig. 7, A to D). Cmpd101 prevented recruitment of GRK2 to the activated MOR, consequently compromising β-arrestin recruitment and receptor internalization. Binding of Cmpd101 to GRK2 induces small conformational changes that stabilize GRK2 in a nonactive, noncatalytic conformation (15). Thus, our results suggest that the unblocked conformation of GRK2/3 is required for the recruitment and interaction of this kinase with MOR.

Although the molecular mechanisms underlying the development of morphine-induced tolerance remain controversial, MOR desensitization of VGCC and GIRK channels is generally accepted to be an important step. As mentioned above, our results suggest that MOR desensitization by high-efficacy ligands has two components: a sustained component, which requires GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component independent of receptor phosphorylation (Fig. 8, A to D, and fig. S5, C and D). This desensitization component, independent of kinase activity, highlights the potential dual role of GRKs, acting as scaffolding/sequestering proteins in addition to their kinase activity, as earlier suggested by Raveh et al. (21). In addition to GRKs, PKC has also been suggested to participate in the development of tolerance to morphine (25), although the molecular basis of this remains unclear. Our patch-clamp studies suggest that, in the STANT-3A MOR mutant, PKC becomes important in mediating ME desensitization (Fig. 8C). Whether PKC phosphorylates Ser363 and/or Thr370 as previously suggested (32, 33) or whether PKC is the soluble factor mediating sustained desensitization remains to be investigated.

In summary, our results demonstrate the complex role of MOR phosphorylation in receptor desensitization and in the recruitment of regulatory and scaffolding proteins. These receptor-effector interactions are likely to participate in the control of physiological opioid effects such as tolerance and dependence.

MATERIALS AND METHODS

Reagents

Morphine HCl was from GlaxoSmithKline or Merck Pharma. The rabbit polyclonal phosphosite-specific antibodies anti-pSer356/pThr357 {4879}, anti-pThr370 {3196}, anti-pSer375 {2493}, anti-pThr376 {3723}, and anti-pThr379 {3686} were generated and extensively characterized previously ({numbers} indicate internal inventory numbers) (10, 11, 13). The phosphorylation-independent rabbit monoclonal anti-MOR antibody {named UMB-3} was obtained from Abcam-Epitomics (34). Secondary antibodies (raised in donkey) conjugated to Alexa Fluor 488, 568, or 647 were from Jackson ImmunoResearch. Coelenterazine h was from NanoLight. Cmpd101 was from Hello Bio. DAMGO, ME, calphostin C, amphotericin B, and M2–anti-FLAG were from Sigma-Aldrich.

Plasmids

For BRET experiments, β-arrestin1–YFP and β-arrestin2–YFP were provided by M. Caron (Duke University, North Carolina), GRK2-Venus was from D. Jensen (Columbia University, New York), and Rab5a-Venus has been previously described (35). GRK2-WT and GRK2-DN (GRK2-K220R) were from M. Smit (Vrije Universiteit, Amsterdam). The phosphorylation-deficient FLAG-MOR mutants and FLAG-MOR-RLuc8 were purchased from GeneArt. For FRET experiments, GRK2-mTurquoise and GRK2-YFP have been previously described (36). β-Arrestin2–mTurquoise was constructed by replacing the CFP coding sequence in β-arrestin2–CFP (37) with mTurquoise (38). For β-Gal complementation experiments, we adapted the plasmids according to PathHunter (DiscoverX Patent: WO 2010042921 A1, US 20100120063 A1). The plasmids were generated through artificial gene synthesis by Eurofins Genomics and cloned into pcDNA3.1. Briefly, the coding sequence for an N-terminal HA tag was added to the WT MOR and the S/T mutant sequences, whereas the C-terminal end was fused with a Gly-rich linker (GGGGSGGGGS) and a short β-Gal fragment (1 to 44 amino acids). All acceptor plasmids, GRKs, and β-arrestins had the larger β-Gal synthetic sequence fragment (45 to 1043 amino acids) fused to their C-terminal end.

siRNA silencing of gene expression

Double-stranded siRNA duplexes with 3′-dTdT overhangs were obtained from Qiagen for GRK2 (5′-AAGAAAUUCAUUGAGAGCGAU-3′), GRK3 (5′-AAGCAAGCUGUAGAACACGUA-3′), and a nonsilencing RNA duplex (5′-GCUUAGGAGCAUUAGUAAA-3′). HEK293 cells were transfected with 150 nM siRNA for single transfection or with 100 nM of each siRNA for double transfection using HiPerFect (Qiagen). Silencing was quantified after 3 days by performing a GRK β-Gal complementation assay. All experiments showed that the siRNA reduced the target protein levels by ≥80% (fig. S4E).

Cell culture

HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% (v/v) fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 95% air and 5% CO2. Transient transfections for BRET experiments were performed using linear polyethylenemine (PEI) with a molecular weight of 25,000 (Polysciences) in a DNA/PEI ratio of 1:6 as previously described (39). For FRET experiments, transient transfections were performed in 6-cm dishes with Effectene (Qiagen) as previously described (36). For transient and stable transfections in β-Gal complementation experiments, TurboFect DNA (Thermo Fisher Scientific) was used. Stably transfected cells were grown in medium supplemented with G418 (400 μg/ml) and/or hygromycin B (100 μg/ml). To increase the total number of HEK293 cells stably expressing MOR or phosphorylation-deficient mutant receptors, we used fluorescence-activated cell sorting (FACS). Trypsinized cells were washed with phosphate-buffered saline (PBS) and transferred into opti-MEM containing an A488-labeled anti-HA antibody at a dilution of 1:1000 (Sigma-Aldrich). After 30-min preincubation at room temperature, cells were centrifuged and the cell pellet was resuspended in FACS buffer [2 mM EDTA, 0.5% (w/v) bovine serum albumin in PBS]. FACS was executed using a BD FACSAria III cell sorter. About 1% of the positive cell population was sorted at an average purity of 85%. Sorted cells were then recultivated. To ensure similar expression levels of WT and mutant receptors, we characterized stable cells using Western blot analysis, cell-surface ELISA assay, and immunocytochemistry.

Patch-clamp experiments were performed in AtT20 cells, which endogenously express GIRK (KirX.3) channels. WT MOR, 354TSST357/A, and 375STANT379/A were all cloned in pcDNA3.0 plasmids with FLAG tag and were expressed stably in AtT20 cells as previously described (13). For patch-clamp experiments, AtT20 cells were seeded on 35-mm polystyrene culture dishes (Becton Dickinson Biosciences) in DMEM containing glucose (4.5 g/liter), penicillin-streptomycin (100 μL/ml), G418 (50 mg/ml), and 10% FBS (50 mg/ml). Cell cultures were maintained in a humidified 5% CO2 atmosphere at 37°C. Cells were ready for recording after 24 hours.

Bioluminescence resonance energy transfer

HEK293 cells were transiently transfected in a 10-cm dish with 1 μg of donor (RLuc8-tagged MOR WT or phosphorylation-deficient mutant) and 4 μg of acceptor (β-arrestin1–YFP, β-arrestin2–YFP, GRK2-Venus, or Rab5a-Venus). For cAMP experiments, cells were transiently transfected with the cAMP sensor using YFP-Epac-RLuc (CAMYEL) BRET biosensor (2.5 μg of FLAG-MOR and 2.5 μg of CAMYEL biosensor per dish). For GRK2 expression experiments, cells were transfected with an additional 2 μg of GRK2-WT, GRK2-DN, or pcDNA3 as a control. After 24 hours, cells were replated into poly-d-lysine–coated white opaque 96-well plates (CulturPlate, PerkinElmer) and allowed to adhere overnight. BRET experiments were performed 48 hours after transfection. Cells were washed with Hanks’ balanced salt solution (HBSS) and equilibrated in HBSS for 30 min at 37°C before the experiment. Coelenterazine h was added to a final concentration of 5 μM 10 min before dual-fluorescence/luminescence measurement in a LUMIstar Omega plate reader (BMG LabTech). The BRET signal was calculated as the ratio of light emitted at 530 nm by YFP or Venus over the light emitted at 430 nm by RLuc8. For concentration-response curves, cells were stimulated with DAMGO (10–10 to 10–4 M) or morphine (10–10 to 10–4 M) for 10 min before BRET measurements. For short kinetic experiments (β-arrestin and GRK2 recruitment), the baseline BRET ratio was measured for five cycles; then, vehicle [0.01% (v/v) dimethyl sulfoxide (DMSO)], DAMGO (1 μM), or morphine (1 μM) was added to the cells, and the BRET signal was measured for 30 min. For longer kinetic experiments (Rab5a), cells were stimulated with vehicle [0.01% (v/v) DMSO], DAMGO (1 μM), or morphine (1 μM) at different time points as stated over an interval of 100 min.

Förster resonance energy transfer

To measure the interaction between MOR and β-arrestins, HEK293T cells were transfected with 0.8 μg of MOR-YFP (or mutants), 0.4 μg of human GRK2, and 0.8 μg of β-arrestin2–mTurquoise. To measure the interaction between MOR and GRK2, HEK293T cells were transfected with 0.5 μg of MOR-YFP (or mutants), 0.5 μg of rat Gɑi1, 0.5 μg of human Gβ1, 0.4 μg of murine Gγ2, and 0.5 μg of GRK2-mTurquoise. On the next day, cells were seeded on round 25-mm poly-d-lysine–coated coverslips, and 48 hours after transfection, FRET was measured as previously described (36), except that a light-emitting diode (LED) excitation system (pE-2; CoolLED) was used for all experiments. FRET traces were not corrected for bleaching effects.

β-Gal complementation

HEK293 cells stably expressing MOR or mutant MOR constructs C-terminally fused with a β-Gal enzyme fragment (β-Gal1–44) and stably or transiently expressing β-arrestins or GRKs fused to an N-terminal deletion mutant of β-Gal (β-Gal45–1043) were used (DiscoverX Patent: WO 2010042921 A1, US 20100120063 A1). β-Arrestin or GRK recruitment results in complementation of the two β-Gal fragments that generate an active enzyme. Thus, levels of active enzyme are a direct result of MOR activation and are quantitated using chemiluminescent β-Gal Juice-plus detection reagents (PJK GmbH) containing the β-Gal substrate. The assay was performed as follows: cells were plated into poly-l-lysine–coated 48-well plates at a density of 125,000 cells per well in medium supplemented with G418 (400 μg/ml) and incubated overnight. After 48 hours, test compounds were prepared at each concentration in DMEM and added to the cells. After a 1-hour incubation at 37°C, a cell lysis reagent [1 M NaH2PO4·H2O, 1 M Na2HPO4·2 H2O, and 0.1% (v/v) Triton X-100 (pH 7.4)] was added to each well. Luminescence was measured 1 hour after detection reagent addition (β-Gal Juice-plus) using a FlexStation III (Molecular Devices, 500-ms integration time).

Western blot

Cells were seeded onto poly-l-lysine–coated 60-mm dishes and grown to 80% confluence. After treatment with agonist, cells were lysed in detergent buffer [50 mM tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10 mM disodium pyrophosphate, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS] in the presence of protease and phosphatase inhibitors (Complete mini and PhosSTOP, Roche Diagnostics). Glycosylated proteins were partially enriched using wheat-germ lectin agarose beads as described (40, 41). Proteins were eluted from the beads using SDS sample buffer for 20 min at 42°C. Samples were split and resolved on 8% SDS–polyacrylamide gels, and after electroblotting, membranes were incubated with anti-pSer356/pThr357 {4879}, anti-pThr370 {3196}, anti-pSer375 {2493}, anti-pThr376 {3723}, or anti-pThr379 {3686} antibodies followed by detection using an enhanced chemiluminescence detection system (Thermo Fisher Scientific). Blots were stripped and incubated again using the phosphorylation-independent anti-MOR antibody {named UMB-3} (34) to ensure equal loading of the gels.

Enzyme-linked immunosorbent assay

HEK293 cells were transfected with 5 μg of DNA (pcDNA3 as a control, FLAG-MOR WT, or phosphorylation-deficient mutants, in 10-cm dishes), replated into poly-d-lysine–coated 48-well plates after 24 h, and allowed to adhere overnight. After 48 hours, cells were fixed with 3.7% (v/v) paraformaldehyde in tris-buffered saline (TBS) for 30 min. For total expression, cells were permeabilized by 30-min incubation with 0.5% (v/v) NP-40 in TBS. Cells were then incubated in blocking buffer [1% (w/v) skim milk powder in 0.1 M NaHCO3] for 4 hours at room temperature and incubated with mouse M2–anti-FLAG antibody (1:2000, overnight at 4°C). After washing three times with TBS, cells were incubated with anti-mouse horseradish peroxidase–conjugated antibody (1:2000) for 2 hours at room temperature. Cells were washed and stained using the SIGMAFAST OPD substrate (Sigma-Aldrich). Absorbance at 490 nm was measured using an EnVision Multilabel Reader (PerkinElmer). Data were normalized to intact HEK293 cells transfected with MOR WT. To measure MOR internalization, HEK293 cells stably expressing the HA-tagged MOR WT, MOR TSST-4A, MOR S375A, MOR TREHPSTANT-4A, or MOR 11S/T-A were preincubated with anti-HA antibody and stimulated with 10 μM DAMGO for 30 min. Receptor sequestration, quantified as the percentage of residual cell-surface receptors in agonist-treated cells, was measured by ELISA as described above.

Confocal imaging and quantitative analysis of MOR internalization

Stably transfected cells were grown on poly-l-lysine–coated coverslips overnight. Cells were then incubated with primary antibody (rabbit anti-HA) in serum-free medium for 2 hours at 4°C. After agonist exposure, cells were fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 30 min at room temperature and washed several times with PBS. Specimens were permeabilized and then incubated with an Alexa Fluor 488–conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology). Specimens were mounted and examined using a Zeiss LSM 510 META laser scanning confocal microscope. For quantitative internalization assays, cells were seeded onto 24-well plates. On the next day, cells were preincubated with anti-HA antibody for 2 hours at 4°C. Cells were then exposed to agonist at 37°C, fixed, and developed with peroxidase-conjugated secondary antibody as described (42, 43).

Electrophysiology

Patch-clamp recordings were performed as previously described (13). Pipettes were pulled from borosilicate glass (A-M Systems) yielding input resistances between 3.5 and 4.5 megohms. For perforated patch-clamp recording, pipettes were filled with internal solution containing 135 mM potassium gluconate, 3 mM MgCl2, and 10 mM Hepes (adjusted to pH 7.4 with KOH). The recording electrodes were first front-filled with this internal solution and then backfilled with the same solution containing amphotericin B (200 μg/ml; in 0.8% DMSO). For whole-cell recording, internal solution contained 135 mM K gluconate, 8 mM NaCl, 8 mM Hepes, 0.5 mM EGTA, 3 mM Mg adenosine 5′-triphosphate, and 0.5 mM Na2 guanosine 5′-triphosphate (pH 7.3). Cells were initially superfused with external bath solution containing 140 mM NaCl, 3 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 10 mM Hepes, and 10 mM glucose (pH 7.4). For measuring IGIRK, the KCl concentration in the bath was increased to 20 mM (substituted for NaCl) before the start of the measurements and was maintained throughout the experiments as previously described by Yousuf et al. (13). Liquid junction potential was calculated to be +16 mV and was adjusted before the start of each recording. Currents were recorded at 37°C in a fully enclosed, temperature-controlled recording chamber using an Axopatch 200B amplifier and pCLAMP 9.2 software and digitized using Digidata 1320 (Axon Instruments, Molecular Devices). Currents were sampled at 100 Hz, low-pass–filtered at 50 Hz, and recorded on hard disk for later analyses. IGIRK was recorded using a 200-ms voltage step to −120 mV from a holding potential of −60 mV delivered every 2 s. Drugs were perfused directly onto cells using a ValveLink 8.2 pressurized pinch valve perfusion system (AutoMate Scientific). In all recorded cells, solution exchange reached steady state within 200 ms (usually within 100 ms), which was confirmed by examination of the current produced at −60 mV by switching from low (3 mM) to high (20 mM) K+ solution.

All data points are plotted as chord GIRK conductance (GGIRK, nS) using the following calculation: [IGIRK (−60 mV) − IGIRK (−120 mV)] pA/60 mV. The extent of MOR desensitization as a percentage was calculated using the following formula: (Post GME/Pre GME) × 100, where “Pre GME” and “Post GME” are the GGIRK increase induced by a submaximal probe concentration of ME (10 nM; see fig. S6), averaged for 4 to 5 points during the peak response (colored points in figures), immediately before compared to 1 min after termination of exposure to a supramaximal concentration of agonist (10 μM ME or 10 μM morphine). Desensitization for different MOR mutants was analyzed using a two-factor ANOVA followed by appropriate post hoc tests as indicated.

Statistics

Data are presented as the mean ± SEM of n ≥ 3 independent experiments. Differences were assessed with GraphPad Prism using one-way or two-way ANOVA followed by Bonferroni-adjusted post hoc tests for multiple comparisons.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/11/539/eaas9609/DC1

Fig. S1. Expression and function of MOR phosphorylation-deficient mutants.

Fig. S2. Inhibition of β-arrestin2 recruitment upon addition of naloxone and lack of internalization by morphine.

Fig. S3. β-Arrestin1 recruitment and MOR internalization upon overexpression of GRK2 WT or GRK2 DN.

Fig. S4. GRK3 recruitment to MOR WT and mutants, inhibition of GRK2 recruitment upon addition of naloxone, kinetics of GRK recruitment, and siRNA controls.

Fig. S5. Fast desensitization component of MOR.

Fig. S6. Patch-clamp traces for corresponding MOR mutants.

Table S1. Potency and maximal response of MOR mutants in cAMP assay.

REFERENCES AND NOTES

Acknowledgments: We thank A. Stewart for technical assistance. Funding: This work was supported by a Deutsche Forschungsgemeinschaft grant (SFB/TR166-TPC5) to S.S., a Monash Fellowship to M.C., a National Health and Medical Research Council (NHMRC) RD Wright Fellowship to M.L.H. (1061687), and an NHMRC project grant (1121029) to M.C. and M.L.H. Author contributions: E.M. performed and analyzed the data of β-Gal experiments; A.B.G. performed and analyzed the data of BRET experiments; A.Y. performed and analyzed electrophysiology experiments; R.S. performed and analyzed the data of some β-Gal experiments; N.M., Y.Y., and M.G. performed and analyzed the data of FRET experiments; J.G.R. generated FRET constructs/reagents; M.B. and C.K. supervised FRET experiments; M.J.C. supervised electrophysiology experiments; M.L.H. and M.C. supervised BRET experiments; and S.S. supervised β-Gal experiments. E.M., A.B.G., and M.C. wrote the manuscript. All authors reviewed the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
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