Research ArticleNeuroscience

G protein signaling–biased agonism at the κ-opioid receptor is maintained in striatal neurons

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Science Signaling  07 Aug 2018:
Vol. 11, Issue 542, eaar4309
DOI: 10.1126/scisignal.aar4309
  • Fig. 1 Divergence in signaling across cell-based KOR signaling assays.

    (A) Membrane [35S]GTPγS binding assay in hKOR-CHO cells treated with triazole 1.1 (blue squares) or iso 2.1 (purple triangles), relative to those treated with U69,593 (red circles) at the indicated doses for 1 hour at room temperature. (B) β-Arrestin2 recruitment assay [enzyme fragment complementation (EFC)] in the DiscoveRx PathHunter U2OS cell line treated with the KOR agonists for 90 min at 37°C. (C and D) Inhibition of forskolin-stimulated cAMP accumulation assay without (C) or with (D) pertussis toxin (PTX) overnight pretreatment in hKOR-CHO cells incubated with the KOR agonists for 30 min at room temperature. (E and F) Intracellular calcium mobilization without or with pertussis toxin pretreatment in hKOR-CHO cells with the KOR agonist treatment. (G) [35S]GTPγS binding assay using membranes prepared from pertussis toxin–pretreated CHO-hKOR cells. (H) Effect of pertussis toxin overnight pretreatment on β-arrestin2 recruitment (EFC) [EC50 (nM) ± SEM: 105 ± 11 (U69), 2980 ± 741 (triazole 1.1), and 3132 ± 894 (iso 2.1); compare to “-PTX” condition in Table 1]. P > 0.05 by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test. (I) Measurement of GIRK channel activation by a Fluorescent Imaging Plate Reader (FLIPR) potassium assay in CHO-hKOR-hGIRK1/2 cells incubated with increased concentrations of the KOR agonists. Data are presented as means ± SEM from n ≥ 3 independent experiments for all assays.

  • Fig. 2 Calculation of ligand bias for across cell-based KOR signaling assays.

    Bias factors of triazole 1.1 (blue boxes) and iso 2.1 (purple boxes) for the indicated pathways are calculated from the transduction coefficients listed in Tables 1 and 2 and are plotted on a log scale. Bias factor for the reference agonist U69,593 is 1 in all assays. Data are presented as means ± SEM. Errors were calculated by unpaired t test comparing ΔLogR values of the test compounds between two functional assays. Signaling pathways are presented as follows: [35S]GTPγS binding [guanosine 5′-triphosphate (GTP)], β-arrestin2 recruitment [βarr2 (EFC)], inhibition of forskolin-stimulated cAMP accumulation (cAMP), intracellular calcium mobilization (Ca2+), and GIRK channel activation (GIRK).

  • Fig. 3 Evaluation of forskolin-stimulated cAMP accumulation in isolated cell membranes.

    (A) Membrane-based inhibition of forskolin-stimulated cAMP accumulation assays using cell membranes prepared from CHO-hKOR cells that were incubated with increased concentrations of test compounds for 30 min at room temperature. Data are presented as means ± SEM of n = 7 independent experiments. (B) Bias factors are calculated using the transduction coefficients listed in Tables 1 and 3 and are plotted on a log scale. Data are presented as means ± SEM. Errors were calculated by unpaired t test comparing ΔLogR values of the test compounds between two functional assays. [35S]GTPγS binding (GTP), cell-based inhibition of forskolin-stimulated cAMP accumulation (cAMP), and membrane-based inhibition of forskolin-stimulated cAMP accumulation (m-cAMP).

  • Fig. 4 Evaluation of bias in a neuronal cell line expressing hKOR (SH-SY5Y-hKOR).

    (A) [35S]GTPγS binding assay using membranes prepared from SH-SY5Y-hKOR cells that were incubated with increased concentrations of test compounds. Graphs are presented as means ± SEM of n ≥ 3 independent experiments. EC50 (nM) ± SEM: 66 ± 11 (U69), 99 ± 22 (triazole 1.1), and 174 ± 67 (iso 2.1); P > 0.05 for triazole 1.1 and iso 2.1 versus U69,593, one-way ANOVA with Bonferroni’s post hoc test. Emax (%) ± SEM: 100 (U69), 92 ± 2 (triazole 1.1), 92 ± 3 (iso 2.1). (B) Concentration response curves of the forskolin-stimulated cAMP accumulation assay in KOR agonist–treated SH-SY5Y cells with or without hKOR overexpression. EC50 (nM) ± SEM: 185 ± 50 (U69), 410 ± 76 (triazole 1.1), and 331 ± 55 (iso 2.1); P < 0.05 for triazole 1.1 versus U69 and P > 0.05 for iso 2.1 versus U69, one-way ANOVA with Bonferroni’s post hoc test. Emax (%) ± SEM: 100 (U69), 108 ± 6 (triazole 1.1), and 116 ± 9 (iso 2.1). Data are presented as means ± SEM of n ≥ 3 independent experiments.

  • Fig. 5 Evaluation of KOR-mediated G protein signaling in striatum and striatal neurons.

    (A) [35S]GTPγS binding assay using membranes prepared from adult mouse striatum tissue. n = 6 independent experiments using at least one mouse striatum tissue each time. (B) Concentration response curves of inhibition of forskolin-stimulated cAMP accumulation in mouse primary striatal neurons. n = 4 independent experiments. (C) Inhibition of cAMP accumulation in response to vehicle (Veh) or KOR agonists (10 μM) without pertussis toxin pretreatment (left of the dashed line) or with pertussis toxin pretreatment (right of the dashed line) in mouse primary striatal neurons. Data are presented as means ± SEM of n = 4 independent experiments. ****P < 0.001 compared to vehicle treatment, one-way ANOVA with Bonferroni’s post hoc test.

  • Fig. 6 Evaluation of β-arrestin2–dependent KOR internalization in striatal neurons.

    (A) KOR internalization concentration response curves in WT primary striatal neurons treated with U69,593 or triazole 1.1 for 30 to 50 min. F(1,17) = 28.31; P < 0.0001, two-way ANOVA; **P < 0.01, Bonferroni’s post hoc test. Each data point was obtained from the mean value of 6 to 11 confocal images of n ≥ 3 independent primary striatal neuron preparations. (B) Representative confocal images of KOR internalization in WT primary striatal neurons treated as in (A). Twenty-one to 69 images were analyzed for each treatment. Scale bar, 5 μm. (C) Scatterplot analysis of KOR internalization in response to 100 nM of either compound in WT and βarr2-KO primary striatal neurons. **P < 0.01 for U69,593 in WT versus U69,593 in βarr2-KO primary striatal neurons by one-way ANOVA with Bonferroni’s post hoc test. Data are presented as means ± SEM from n ≥ 3 independent experiments.

  • Table 1 Signaling parameters for the KOR agonists in the cell-based functional assays.

    Data are presented as means ± SEM from n ≥ 3 independent experiments performed in duplicate to quadruplicate. Emax values were normalized calculation to the maximum stimulation by U69,593. Ca2+ accumulation, intracellular calcium mobilization assay; K+ accumulation, GIRK channel activation assay.

    Compound[35S]GTPγS binding
    EC50 (nM)Emax (%)LogRΔLogR
    U69,59366 ± 131007.26 ± 0.090
    1.196 ± 1095 ± 17.00 ± 0.05−0.29 ± 0.07
    2.1155 ± 3094 ± 26.80 ± 0.07−0.44 ± 0.06
    βarr2 recruitment (EFC)
    EC50 (nM)Emax (%)LogRΔLogR
    U69,593112 ± 111007.00 ± 0.060
    1.13338 ± 40588 ± 65.44 ± 0.04−1.56 ± 0.09
    2.12447 ± 27682 ± 45.59 ± 0.06−1.42 ± 0.09
    Forskolin-stimulated cAMP accumulation
    EC50 (nM)Emax (%)LogRΔLogR
    U69,59312 ± 21008.02 ± 0.090
    1.1309 ± 6594 ± 56.49 ± 0.12−1.64 ± 0.13
    2.1303 ± 4698 ± 56.55 ± 0.04−1.33 ± 0.09
    Ca2+ accumulation
    EC50 (nM)Emax (%)LogRΔLogR
    U69,59319 ± 51007.79 ± 0.120
    1.11064 ± 18697 ± 95.96 ± 0.14−1.91 ± 0.16
    2.11594 ± 49198 ± 85.97 ± 0.15−1.82 ± 0.19
    K+ accumulation
    EC50 (nM)Emax (%)LogRΔLogR
    U69,5938 ± 21008.29 ± 0.180
    1.121 ± 8108 ± 88.03 ± 0.27−0.23 ± 0.03
    2.122 ± 6105 ± 97.85 ± 0.13−0.41 ± 0.18
  • Table 2 Analysis of bias factors between functional assays.

    Using U69,593 as a reference agonist, the ΔΔLogR and bias factors (10ΔΔlogRassay1-assay2) were calculated as described in “Data analysis and statistics” (Materials and Methods) using the ΔLogR values listed in Table 1. GTP, [35S]GTPγS binding assay; βarr2, β-arrestin2 recruitment assay (EFC); cAMP, forskolin-stimulated cAMP accumulation assay; Ca2+, intracellular calcium mobilization assay; GIRK, GIRK channel activation assay; 95% CI, 95% confidence interval.

    Comparison of assays1.12.1
    ΔΔLogR (95% CI)Bias factorΔΔLogR (95% CI)Bias factor
    GTP/βarr21.27 (1.03 to 1.52)190.98 (0.73 to 1.22)9
    GTP/cAMP1.35 (1.02 to 1.69)230.89 (0.64 to 1.13)8
    GTP/Ca2+1.62 (1.24 to 1.99)421.38 (0.94 to 1.82)24
    GTP/GIRK−0.06 (−0.30 to 0.18)0.9−0.04 (−0.39 to 0.32)0.9
    cAMP/GTP−1.35 (−1.69 to −1.02)0.04−0.89 (−1.13 to −0.64)0.1
    cAMP/βarr2−0.08 (−0.46 to 0.30)0.80.09 (−0.20 to 0.38)1
    cAMP/Ca2+0.26 (−0.21 to 0.74)20.49 (0.03 to 0.95)3
    cAMP/GIRK−1.41 (−1.88 to −0.94)0.04−0.92 (−1.34 to -0.50)0.1
    Ca2+/GTP−1.62 (−1.99 to −1.24)0.02−1.38 (−1.82 to −0.94)0.04
    Ca2+/βarr2−0.35 (−0.77 to 0.08)0.5−0.40 (−0.91 to 0.10)0.4
    Ca2+/cAMP−0.26 (−0.74 to 0.21)0.5−0.49 (−0.95 to -0.03)0.3
    Ca2+/GIRK−1.68 (−2.21 to −1.14)0.02−1.41 (−2.12 to −0.71).0.03
    GIRK/GTP0.06 (−0.18 to 0.03)1.10.04 (−0.32 to 0.39)1
    GIRK/βarr21.33 (1.04 to 1.62)211.01 (0.57 to 1.46)10
    GIRK/cAMP1.41 (0.94 to 1.88)260.92 (0.50 to 1.34)8
    GIRK/Ca2+1.68 (1.14 to 2.21)481.41 (0.71 to 2.12)26
  • Table 3 Signaling parameters for forskolin-stimulated cAMP accumulation in CHO-hKOR cells or using CHO-hKOR cell membranes.

    U69,593 serves as the reference agonist. Data are presented as means ± SEM from n ≥ 4 independent experiments performed in duplicate to quadruplicate. P values were obtained by comparing the values of the KOR agonist treatment in CHO-hKOR cells to CHO-hKOR cell membranes using a Student’s t test.

    Assay systemU69,5931.1Fold difference (EC50 1.1/EC50 U69)LogRΔLogR
    hKOR (cell)*EC50 (nM)12 ± 2309 ± 6534 ± 98.02 ± 0.090
    Emax (%)10094 ± 56.49 ± 0.12−1.64 ± 0.13
    hKOR (membrane)EC50 (nM)35 ± 8155 ± 305 ± 0.67.54 ± 0.110
    Emax (%)10098 ± 46.97 ± 0.14−0.57 ± 0.15

    *Whole-cell forskolin-stimulated cAMP accumulation assay in CHO-hKOR cells was shown in Table 1 for comparison to the membrane-based forskolin-stimulated cAMP accumulation.

    P < 0.01.

    P < 0.05

    • Table 4 Bias analysis between [35S]GTPγS binding and forskolin-stimulated cAMP accumulation for SH-SY5Y-hKOR cells.

      LogR, ΔLogR, ΔΔLogR, and bias factors (10ΔΔlogRassay1-assay2) were calculated using U69,593 as the reference agonist, as described in “Data analysis and statistics” using the values stated in the figure legends of Fig. 4. Data are presented as means ± SEM from n ≥ 3 independent experiments performed in duplicate to triplicate. G, [35S]GTPγS; cAMP, forskolin-stimulated cAMP accumulation; 95% CI, 95% confidence interval.

      Compound[35S]GTPγS bindingcAMPΔΔLogR (95% CI)Bias factor (G/cAMP)
      LogRΔLogRLogRΔLogR
      U69,5937.18 ± 0.0606.32 ± 0.30001
      1.17.06 ± 0.15−0.112 ± 0.096.26 ± 0.30−0.059 ± 0.12−0.053(−0.43 to 0.32)0.9
      2.16.74 ± 0.13−0.434 ± 0.086.14 ± 0.48−0.219 ± 0.06−0.215 (−0.46 to 0.03)0.6
    • Table 5 Signaling parameters for the assays using mouse striatal membranes and mouse primary striatal neurons.

      U69,593 serves as the reference agonist. Data are presented as means ± SEM from n ≥ 4 independent experiments. G, [35S]GTPγS binding; cAMP, forskolin-stimulated cAMP accumulation; KORint, KOR internalization; 95% CI, 95% confidence interval.

      Compound[35S]GTPγS bindingΔΔLog(Emax/EC50) (G/cAMP, 95% CI)Bias factor (G/cAMP)
      EC50 (nM)Emax (%)Log(Emax/EC50)ΔLog(Emax/EC50)
      U69221 ± 261008.68 ± 0.06001
      1.1486 ± 98*141 ± 538.51 ± 0.07−0.17 ± 0.03−0.02 (−0.25 to 0.10)1.0
      CompoundInhibition of cAMP accumulationΔΔLog(Emax/EC50) (G/KORint)Bias factor (G/KORint)
      EC50 (nM)Emax (%)Log(Emax/EC50)ΔLog(Emax/EC50)
      U6917 ± 41007.76 ± 0.09001
      1.145 ± 12167 ± 207.57 ± 0.15−0.19 ± 0.11.3724
      CompoundKOR internalizationΔΔLog(Emax/EC50) (cAMP/KORint)Bias factor (cAMP/KORint)
      EC50 (nM)Emax (%)Log(Emax/EC50)ΔLog(Emax/EC50)
      U69191009.73001
      1.1238378.19−1.541.3623

      *P < 0.05, Student’s t test for U69,593 versus triazole 1.1.

      †Values were obtained from the concentration response curve of which each data point is from the average of n ≥ 3 independent neuron preparations.

      Supplementary Materials

      • www.sciencesignaling.org/cgi/content/full/11/542/eaar4309/DC1

        Fig. S1. KOR agonists do not stimulate cAMP accumulation.

        Fig. S2. KOR agonist potency for inhibiting forskolin-stimulated cAMP accumulation is not affected by changing the incubation time.

        Fig. S3. RGS protein effects on KOR-regulated adenylyl cyclase activity.

        Fig. S4. Triazole 1.1 and iso 2.1 display similar signaling profiles in CHO and U2OS cells stably expressing mouse KOR as they do expressing human KOR.

        Fig. S5. β-arrestins are not required for KOR-regulated adenylyl cyclase activity.

        Fig. S6. β-arrestins are required for KOR internalization.

        Table S1. Signaling parameters for the time course inhibition of forskolin-stimulated cAMP accumulation in CHO-hKOR cells.

        Table S2. Signaling parameters for the KOR agonists in the functional assays in CHO-mKOR cells.

        Table S3. Signaling parameters for the KOR agonists in the inhibition of forskolin-stimulated cAMP accumulation in WT and β-arrestin1/2-KO MEF-mKOR cells.

        Table S4. Signaling parameters for the inhibition of cAMP accumulation in CHO cells stably expressing hKOR alone or with hRGS4, hRGS9.2, or hRGS12.3.

        Table S5. Primer sequences.

      • This PDF file includes:

        • Fig. S1. KOR agonists do not stimulate cAMP accumulation.
        • Fig. S2. KOR agonist potency for inhibiting forskolin-stimulated cAMP accumulation is not affected by changing the incubation time.
        • Fig. S3. RGS protein effects on KOR-regulated adenylyl cyclase activity.
        • Fig. S4. Triazole 1.1 and iso 2.1 display similar signaling profiles in CHO and U2OS cells stably expressing mouse KOR as they do expressing human KOR.
        • Fig. S5. β-arrestins are not required for KOR-regulated adenylyl cyclase activity.
        • Fig. S6. β-arrestins are required for KOR internalization.
        • Table S1. Signaling parameters for the time course inhibition of forskolin-stimulated cAMP accumulation in CHO-hKOR cells.
        • Table S2. Signaling parameters for the KOR agonists in the functional assays in CHO-mKOR cells.
        • Table S3. Signaling parameters for the KOR agonists in the inhibition of forskolin-stimulated cAMP accumulation in WT and β-arrestin1/2-KO MEF-mKOR cells.
        • Table S4. Signaling parameters for the inhibition of cAMP accumulation in CHO cells stably expressing hKOR alone or with hRGS4, hRGS9.2, or hRGS12.3.
        • Table S5. Primer sequences.

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