Research ArticlePharmacology

Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

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Science Signaling  26 Mar 2019:
Vol. 12, Issue 574, eaau8072
DOI: 10.1126/scisignal.aau8072
  • Fig. 1 Characterization of phosphosite-specific NOP receptor antibodies.

    (A) Schematic representation of the hNOP receptor (hNOPR). All potential intracellular phosphate acceptor sites are indicated (gray). Ser346, Ser351, and Thr362/Ser363 were targeted for the generation of phosphosite-specific antibodies, and the epitope used for generating a phosphorylation-independent antibody (NOP receptor) is indicated by a black line. (B) HEK293 cells were transiently transfected with either the wild-type hNOP receptor or its 6S/T-A mutant. After 48 hours, cells were labeled with carrier-free [32P]orthophosphate (200 μCi/ml). Labeled cells were either not treated (−) or treated (+) with 10 μM N/OFQ for 10 min, and whole-cell receptor phosphorylation was determined by SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. (C) Dot-blot analysis on serial dilutions of peptides P1 to P4 to characterize antibody to pThr362/pSer363. (D and E) Characterization of phosphosite-specific antibodies directed against Ser346, Ser351, or Thr362/Ser363 using λ-phosphatase. HEK293 cells stably expressing the HA-tagged hNOP receptor were either not treated (−) or treated (+) with 10 μM N/OFQ for 10 min. Lysates were then either incubated (+) or not (−) with λ-phosphatase and immunoblotted with the phosphosite-specific antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed with the phosphorylation-independent antibody to the NOP receptor as a loading control. In all panels, blots are representative of one of three independent experiments. Molecular mass markers (in kDa) are indicated (left).

  • Fig. 2 Characterization of phosphosite-specific NOP receptor antibodies using receptor mutants.

    (A) Sequence of the C-terminal tail of the hNOP receptor showing all potential phosphate acceptor sites. Serine (S) and threonine (T) residues depicted in gray were exchanged to alanine. (B and C) HEK293 cells stably expressing HA-tagged hNOP, S337A/S346A/S351A and T362A/S363A/T365A, S363A, 6S/T-A, or NOP-10S/T-A were either not treated (−) or treated (+) with 10 μM N/OFQ for 10 min, and lysates were then immunoblotted with the antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed with antibodies to the NOP receptor or HA-tag. Blots are representative of n = 3 independent experiments.

  • Fig. 3 Time course of N/OFQ-induced Ser346, Ser351, and Thr362/Ser363 phosphorylation and internalization.

    (A to D) HEK293 cells stably expressing hNOP receptors were exposed to 10 μM N/OFQ for the indicated times and temperatures {(A) and (B): up to 60 min at 37°C; (C) and (D): up to 20 min at 22°C [room temperature (RT)]}, and lysates were immunoblotted with the indicated antibodies. Blots are representative of n = 5 (A and C) or 6 (B and D) independent experiments. Blots were stripped and reprobed for the NOP receptor. (E) HEK293 cells stably expressing the HA-tagged hNOP receptor were preincubated with antibody to HA-tag and subsequently exposed to 10 μM N/OFQ for up to 60 min at 22°C. Cells were fixed, permeabilized, immunofluorescently stained, and examined using confocal microscopy. Images are representative of one of three independent experiments. Scale bar, 20 μm. (F) Stably HA-tagged hNOP receptor–expressing HEK293 cells were preincubated with antibody to HA-tag and stimulated with 10 μM N/OFQ for up to 90 min at 37°C. Cells were then fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells. Data are means ± SEM of six independent experiments performed in quadruplicate.

  • Fig. 4 NOP receptor phosphorylation is mediated by GRK2 and GRK3.

    (A) HEK293 cells stably expressing the hNOP receptor were preincubated with either vehicle [dimethyl sulfoxide (DMSO); Control] or the GRK2/3-specific inhibitor compound 101 (Cmpd 101) at 30 μM for 30 min at 37°C and then exposed to 10 μM N/OFQ, 10 μM forskolin, or 1 μM PMA (or not, −) for 10 min. Lysates were then immunoblotted with antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed for the NOP receptor. Blots are representative of n = 3 independent experiments. (B) Cells described in (A) were preincubated with vehicle [DMSO, (−)] or compound 101 at the indicated concentrations for 30 min at 37°C and then treated with water (−) or 10 μM N/OFQ for 10 min at 37°C. Lysates were blotted as described in (A). Blots are representative of n = 4 independent experiments. (C and D) Cells described in (A) were transfected with either siRNA targeting GRK2, GRK3, or GRK2 and GRK3 (GRK2/3) or a control (SCR) for 72 hours (C) or with GRK2 or GRK3 expression plasmids or an empty vector (MOCK) for 48 hours (D) and then stimulated with 10 μM N/OFQ for 10 min at 37°C. Lysates were immunoblotted with antibody to pThr362/Ser363. Blots were stripped and reprobed for the NOP receptor. Densitometry, above the blots, was normalized to those in SCR- or MOCK-transfected cells, which were set to 100%. Data are means ± SEM from five to six independent experiments. *P < 0.05 versus SCR or MOCK by one-way analysis of variance (ANOVA) with Bonferroni post-test.

  • Fig. 5 Agonist-selective NOP receptor phosphorylation and internalization.

    (A) HEK293 cells stably expressing HA-tagged hNOP receptors were preincubated with HA antibody and then stimulated with 10 μM N/OFQ, Ro64-6198, MCOPPB, SCH221510, NNC 63-0532, AT-202, buprenorphine (BUP), norbuprenorphine (norBUP), cebranopadol, or vehicle (according to the solvent) for 60 min at 22°C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative of n = 3 independent experiments. Scale bar, 20 μm. (B) NOP receptor–expressing HEK293 cells were treated with the compounds listed in (A) (−, vehicle solvent) at concentrations ranging from 10−9 to 10−5 M for 10 min at 37°C, and lysates were immunoblotted with antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed for the NOP receptor. Blots are representative of n = 3 to 5 experiments. (C) HEK293 cells stably expressing hNOP receptors were preincubated with antibody to HA-tag and treated with vehicle (solvent) or 10 μM of the compounds listed in (A) for 60 min at 37°C. Cells were fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells. Data are means ± SEM from 12 independent experiments performed in quadruplicate. (D) Maximum NOP receptor ligand–induced phosphorylation at Thr362/Ser363 from data in (B). Data are means ± SEM from three independent experiments. *P < 0.05 versus N/OFQ by one-way ANOVA with Bonferroni post-test. (E) Correlation between NOP receptor phosphorylation and internalization induced by different ligands in HEK293 cells from data in (A) to (D). Abscissae, ligand-induced internalization in percentage (normalized to N/OFQ); ordinates, ligand-induced phosphorylation in percentage (normalized to N/OFQ); solid line, linear regression of the data points; correlation coefficient r = 0.8581.

  • Fig. 6 G protein signaling of chemically diverse NOP receptor agonists.

    (A to G) Agonist-induced hyperpolarization can be measured by changes in fluorescence intensity of fluorescent oxonol dyes. A reduction of fluorescent signal intensity is indicative of Gβγ protein–mediated GIRK channel activation. AtT-20 cells stably expressing the hNOP receptor were first preloaded with the dye. Thereafter, a baseline is measured for 60 s before cells were stimulated with vehicle (according to the solvent) or with Ro64-6198 (A), MCOPPB (B), SCH221510 (C), NNC 63-0532 (D), AT-202 (E), buprenorphine (F), cebranopadol (G), or N/OFQ (A to G) at a concentration range of 10−6 to 10−13 M for 180 s. Dose-response curves were calculated with OriginPro using sigmoidal nonlinear fitting. Vehicle-induced changes in fluorescence signal (background) were subtracted from signals obtained using agonist-containing solutions. Data are means ± SEM from three independent experiments performed in duplicate. ΔRFU is change in relative fluorescence. (H) Correlation between NOP receptor phosphorylation (pThr362/Ser363) and GIRK channel activation induced by the different ligands, color-coordinated with (A) to (G). Abscissae, ligand-induced G protein activation (EC50 values); ordinates, ligand-induced phosphorylation in percentage (normalized to N/OFQ); solid line, linear regression of the data points; correlation coefficient r = 0.6859.

  • Fig. 7 Antagonist-selective inhibition of N/OFQ-induced phosphorylation, internalization, and G protein signaling.

    (A and B) HEK293 cells stably expressing hNOP receptors were preincubated (+) or not (−) with 50 μM naloxone, J-113397, or SB 612111 for 30 min at 37°C and then treated with vehicle (water, −) or with 10 μM N/OFQ (+) for 10 min at 37°C. Cell lysates were then immunoblotted with antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed for the NOP receptor. Blots are representative of n = 3 independent experiments. (C) HEK293 cells stably expressing the NOP receptor were preincubated with antibody to HA-tag and then treated with vehicle (DMSO), 50 μM naloxone, J-113397, or SB 612111 and with or without 10 μM N/OFQ for 60 min at 37°C. Cells were then fixed and labeled with a peroxidase-conjugated secondary antibody, and receptor internalization was measured by ELISA and quantified as percentage of internalized receptors in agonist-treated cells. Data are means ± SEM from six independent experiments performed in quadruplicate. *P < 0.05 versus N/OFQ by one-way ANOVA with Bonferroni post-test. (D) Cells described and treated as in (C), except treated at 22°C, were fixed, permeabilized, immunofluorescently stained, and examined using confocal microscopy. Images are representative of one of three independent experiments. Scale bar, 20 μm.

  • Fig. 8 Tissue distribution of NOP receptor and nanoLC-MS/MS analysis of NOP receptor phosphorylation in vivo.

    (A) Distribution of NOP receptor in NOP-eGFP knock-in mouse tissue. Anesthetized NOP-eGFP knock-in mice were sacrificed, and tissues were removed. The NOP receptor was then immunoprecipitated from homogenates with anti-GFP (green fluorescent protein) protein agarose beads, and samples were immunoblotted with the phosphorylation-independent antibody to the NOP receptor or antibody to GFP. Bottom, prolonged enhanced chemiluminescence (ECL) detection exposure. Blots are representative of one of three independent experiments. (B) MS coverage of the NOP receptor sequence from mouse brain. The schema represents the secondary structure of the mouse NOP receptor. Filled (blue and black) symbols indicate the protein sequence covered by nanoLC-MS/MS; the phosphorylated residues identified are black. Red circles indicate the trypsin cleavage sites. Pm, plasma membrane; e1 to e3, extracellular loops; i1 to i4, intracellular loops. (C) List of phosphorylated and unphosphorylated NOP receptor peptides identified by nanoLC-MS/MS in the mouse brain. Amino acids belonging to the GFP sequence are in italics. Theo. mass, theoretical mass (in Da); MC, missed cleavage. (D and E) ETD MS/MS spectra of the monophosphorylated peptide 339-EMQVpSDRVR-347 (D) [triply charged precursor ion, MH3+, at mass/charge ratio (m/z) 400.5128] and 359-pT/pSETVPRPAGSIATMVSK-376 (E) (triply charged precursor ion, MH3+, at m/z 637.9797) display series of c- and z-ions, indicating that Ser343 (D) and Thr359 or Ser360 (E) are phosphorylated, respectively. Red labels indicate site-determining ions and the corresponding peaks in the spectrum. Blue labels indicate fragment ions that confirm the site localization and exclude another potential site. pS or pT, phosphorylated serine or threonine residues.

  • Fig. 9 Agonist-selective NOP receptor phosphorylation in mouse brain.

    (A) Schematic representation of the human (h) and mouse (m) NOP receptor. All potential intracellular phosphate acceptor sites are indicated (gray). (B) After intraperitoneal injection of 0.9% NaCl (0) or AT-202 (0.3 to 30 mg/kg) for 30 min, NOP-eGFP knock-in mice were euthanized and brains were removed. The NOP receptor was immunoprecipitated with anti-GFP protein agarose beads and immunoblotted with antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed for the NOP receptor or GFP. Blots are representative of n = 3 independent experiments. (C) As in (B), but mice were treated with SB 612111 or AT-202 singly or pretreated with SB 612111 for 30 min followed by AT-202 (each 30 mg/kg). (D) After intracerebroventricular injection of compound 101 (0.3 to 30 nmol), NOP-eGFP knock-in mice were treated with AT-202 (30 mg/kg for 30 min) and euthanized, and brains were removed. Homogenates underwent immunoprecipitation with anti-GFP agarose beads, and the resulting samples were immunoblotted for the phosphorylated (pThr362/Ser363) NOP receptor. Blots were stripped and reprobed with the GFP antibody as a loading control. Blots are representative of n = 3 independent experiments. (E) As in (B), but mice were treated with 0.9% NaCl (−) or AT-202, Ro64-6198, NNC 63-0532, SCH221510, or MCOPPB (30 mg/kg) for 30 min. (F and G) Imaging (F) and analysis (G) of NOP receptor internalization in ventral midbrain neurons. Primary cultures from NOPR-eYFP mice were treated for 1 hour with 1 μM of the indicated agonist, followed by live cell spinning disk confocal imaging. Images are representative, and data are means ± SEM of over 40 images from at least two dishes per condition. *P < 0.05 versus N/OFQ by one-way ANOVA with Bonferroni post-test.

  • Table 1 NOP receptor peptide sequences used for generation of phosphosite-specific antisera.

    List of peptide sequences used for generating phosphosite-specific antibodies against individual phosphorylated forms of the NOP receptor and a phosphorylation-independent antiserum targeting the NOP receptor at the end of the C-terminal domain. Endogenous cysteines were exchanged (abu).

    Antiserum nameSequence used for
    immunization
    Amino acid
    position in hNOP
    receptor
    Ser346REMQV-(p)S-DRVR341–350
    Ser351DRVR-(p)S-IAKDV347–356
    Thr362/Ser363LG-abu-K-(p)T-(p)S-ETVPR358–368
    NOP receptor
    (phosphorylation
    independent)
    VRSIAKDVGLG-abu-
    KTSETVPRPA
    349–370

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/574/eaau8072/DC1

    Fig. S1. NOP receptor phosphorylation is mediated by GRK2 and GRK3.

    Fig. S2. Correlation between NOP receptor phosphorylation and internalization.

    Fig. S3. Clinically relevant opioids as well as MOP receptor–, delta opioid (DOP) receptor–, and kappa opioid (KOP) receptor–selective agonists failed to induce NOP receptor phosphorylation.

    Fig. S4. Establishment of the membrane potential assay.

    Fig. S5. Quantification of agonist-induced NOP receptor phosphorylation in mouse brain.

  • This PDF file includes:

    • Fig. S1. NOP receptor phosphorylation is mediated by GRK2 and GRK3.
    • Fig. S2. Correlation between NOP receptor phosphorylation and internalization.
    • Fig. S3. Clinically relevant opioids as well as MOP receptor–, delta opioid (DOP) receptor, and kappa opioid (KOP) receptor–selective agonists failed to induce NOP receptor phosphorylation.
    • Fig. S4. Establishment of the membrane potential assay.
    • Fig. S5. Quantification of agonist-induced NOP receptor phosphorylation in mouse brain.

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