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
  • 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.

  • 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).

  • 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).

  • 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).

  • 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.

  • 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.

  • 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).

  • 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).

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

    NA, not available.

    MORβ-Arrestin1β-Arrestin2
    BRETβ-GalBRETβ-Gal
    pEC50Emax
    (% of WT
    DAMGO)
    pEC50Emax
    (% of WT
    DAMGO)
    pEC50Emax
    (% of WT
    DAMGO)
    pEC50Emax
    (% of WT
    DAMGO)
    WTDAMGO6.05 ± 0.061005.82 ± 0.071006.28 ± 0.041005.94 ± 0.03100
    Morphine6.12 ± 0.0533.57 ± 2.18
    TSST-4ADAMGO6.10 ± 0.0691.59 ± 1.565.86 ± 0.0536.33 ± 6.79^6.36 ± 0.0294.08 ± 3.765.58 ± 0.0556.73 ± 7.61^
    Morphine5.85 ± 0.1429.21 ± 4.11
    S375ADAMGO5.89 ± 0.0736.20 ± 6.27^6.71 ± 0.1332.20 ± 5.73^6.30 ± 0.0171.73 ± 6.47^6.08 ± 0.1849.36 ± 16.95^
    Morphine6.37 ± 0.1417.31 ± 0.90^
    STANT-3ADAMGONANA6.20 ± 0.0656.26 ± 1.81^NANA
    MorphineNANA6.40 ± 0.0814.76 ± 1.66^NANA
    TREHPSTANT-4ADAMGO5.95 ± 0.4828.47 ± 7.18^NANA5.72 ± 0.0542.55 ± 6.31^
    MorphineNANA
    11S/T-ADAMGO8.27 ± 1.7423.46 ± 2.33^6.16 ± 0.0241.90 ± 5.48^6.08 ± 0.1743.73 ± 5.16^
    Morphine6.29 ± 0.0815.92 ± 1.67^

    ^Significance compared to WT (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).
      MORBRETβ-Gal
      pEC50Emax
      (% of mock DAMGO)
      pEC50Emax
      (% of mock or SCR)
      MockDAMGO6.50 ± 0.021005.72 ± 0.02100
      Morphine5.95 ± 0.0525.72 ± 2.106.51 ± 0.38100
      GRK2-WT
      OE
      DAMGO8.03 ± 0.04^176.43 ± 8.43^5.81 ± 0.01128.98 ± 17.03
      Morphine7.33 ± 0.05^146.06 ± 6.18^6.16 ± 0.26183.99 ± 28.26
      GRK2-DN
      OE
      DAMGO6.41 ± 0.0362.23 ± 3.18^
      Morphine6.55 ± 0.0212.56 ± 1.66
      GRK3-WT
      OE
      DAMGONANA6.51 ± 0.03157.94 ± 18.66
      MorphineNANA6.05 ± 0.14144.58 ± 40.65
      SCRDAMGONANA5.71 ± 0.13100
      MorphineNANA
      GRK2 KDDAMGONANA6.09 ± 0.1272.39 ± 4.35
      MorphineNANA
      GRK3 KDDAMGONANA6.14 ± 0.1585.64 ± 2.51
      MorphineNANA

      ^Significance compared to WT (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.
        MORBRETβ-Gal
        pEC50Emax
        (% of WT DAMGO)
        pEC50Emax
        (% of WT DAMGO)
        WTDAMGO6.22 ± 0.141006.39 ± 0.12100
        Morphine6.37 ± 0.2037.88 ± 7.226.57 ± 0.36 (0.27)56.13 ± 9.64
        TSST-4ADAMGO6.50 ± 0.1399.02 ± 9.385.72 ± 0.1145.35 ± 1.58^
        Morphine6.47 ± 0.0842.72 ± 11.93
        S375ADAMGO6.08 ± 0.0586.02 ± 3.795.80 ± 0.0940.15 ± 7.32^
        Morphine5.92 ± 0.2730.36 ± 7.33
        STANT-3A/
        TREHPSTANT-4A
        DAMGO5.77 ± 0.1558.33 ± 6.62^6.15 ± 0.3836.73 ± 4.27^
        Morphine5.63 ± 0.5126.77 ± 5.45
        11S/T-ADAMGO5.77 ± 0.0853.33 ± 2.48^6.13 ± 1.4633.86 ± 3.64^
        Morphine5.57 ± 0.0523.20 ± 3.11

        ^Significance compared to WT (P < 0.01) by two-way ANOVA with Dunnett’s multiple comparison test.

        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.

        • This PDF file includes:

          • 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.

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