Journal: Advanced Science

Article Title: EphB1 Receptor Blockade Augments Morphine Antinociception via Inhibiting GRK2‐Mediated µ‐Opioid Receptor Internalization

doi: 10.1002/advs.78099

Figure Lengend Snippet: Spinal administration of ephrinB2‐Fc diminishes morphine antinociception. (A, B) Western blot showing that morphine activated ephrinB2‐EphB1 receptor signaling in DRGs (A) and in the spinal cord (B). Left, representative bands. Right, statistical summary. n = 3 in each group. (C, D) Intrathecal administration of ephrinB2‐Fc diminished the analgesic effect of morphine in the tail‐flick test (C) and hot‐plate test (D). Control IgG‐Fc or ephrinB2‐Fc was intrathecally injected 30 min prior to intrathecal injection of morphine. Left, %MPE. Right, AUC. (E–G) Intrathecal administration of ephrinB2‐Fc diminished the analgesic effects of DAMGO (E), but not DPDPE (F), or U69593 (G), in the tail‐flick test (left) and hot‐plate test (right). Control IgG‐Fc and eprhinB2‐Fc were injected intrathecally 30 min before intrathecal administration of DAMGO, DPDPE, or U69593 . Upper, %MPE. Lower, AUC. (C–G) Sample sizes were indicated in brackets. Statistical analysis by unpaired Student's t test in (A) to (G), or two‐way ANOVA with Bonferroni's multiple comparison test in (C) to (G). n.s., no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: DAMGO (HY‐P0210), DPDPE (HY‐P1334), Paroxetine (HY‐122272), and STA‐013 (HY‐179572) were purchased from MedChemExpress; U69593 (U103) was purchased from Sigma; Recombinant mouse ephrinB2‐Fc (496‐EB‐200) was purchased from R&D Systems; control mouse IgG‐Fc (JAC‐015‐000‐008) was purchased from Jackson ImmunoResearch.

Techniques: Western Blot, Tail Flick Test, Hot Plate Test, Control, Injection, Analysis, Comparison

Journal: Advanced Science

Article Title: EphB1 Receptor Blockade Augments Morphine Antinociception via Inhibiting GRK2‐Mediated µ‐Opioid Receptor Internalization

doi: 10.1002/advs.78099

Figure Lengend Snippet: EphB1 receptors regulate MOR trafficking and control the phosphorylation of MOR at Ser 375 . (A) Schematic illustration of generating Neuro‐2a cells overexpressing MOR. (B) Western blot showing the expression of total protein, plasma membrane (PM) distribution, and cytoplasm distribution of MOR in Neuro‐2a cells overexpressing MOR following treatment of ephrinB2‐Fc (0.01 µg/µL). Left, representative bands. Right, statistical summary. Each experiment was repeated three times. (C) Immunofluorescence staining showing MOR internalization in Neuro‐2a cells overexpressing MOR following treatment of ephrinB2‐Fc (0.01 µg/µL). Left, representative images. The scale bar indicates 10 µm. Right, statistical summary. n = 36 cells in IgG‐Fc + Vehicle group, n = 32 cells in ephrinB2‐Fc + Vehicle group, n = 30 cells in IgG‐Fc + DAMGO group, and n = 47 cells in ephrinB2‐Fc + DAMGO group. (D) Schematic illustration of GRK2‐mediated phosphorylation of MOR at Ser 375 and MOR internalization. MOR agonist induces binding of GRK2, which triggers the phosphorylation of MOR at Ser 375 . β‐arrestin is then recruited and mediates the internalization of MOR. (E) Western blot showing increased phosphorylation of EphB1 receptor and MOR at Ser 375 in Neuro‐2a cells overexpressing MOR following treatment with ephrinB2‐Fc (0.01 µg/µl). Left, representative bands. Right, statistical summary. Each experiment was repeated three times. (F, G) Western blot showing increased phosphorylation of MOR at Ser 375 in DRGs (F) and spinal cord (G) following intrathecal administration of ephrinB2‐Fc (50 ng). Control IgG‐Fc or ephrinB2‐Fc was intrathecally injected 30 min before intrathecal injection of DAMGO or Vehicle. Left, representative bands. Right, statistical summary. n = 3 in each group. (H, I) Western blot showing that knockout of EphB1 receptors in Vglut2 + neurons inhibited DAMGO‐induced phosphorylation of MOR at Ser 375 in DRGs (H) and the spinal cord (I). Left, representative bands. Right, statistical summary. n = 3 in each group. Statistical analysis by unpaired Student's t ‐test in (B), (C), (E), (F), and (G), or one‐way ANOVA with Bonferroni's multiple comparison test in (H) and (I). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: DAMGO (HY‐P0210), DPDPE (HY‐P1334), Paroxetine (HY‐122272), and STA‐013 (HY‐179572) were purchased from MedChemExpress; U69593 (U103) was purchased from Sigma; Recombinant mouse ephrinB2‐Fc (496‐EB‐200) was purchased from R&D Systems; control mouse IgG‐Fc (JAC‐015‐000‐008) was purchased from Jackson ImmunoResearch.

Techniques: Control, Phospho-proteomics, Western Blot, Expressing, Clinical Proteomics, Membrane, Immunofluorescence, Staining, Binding Assay, Injection, Knock-Out, Analysis, Comparison

Journal: Advanced Science

Article Title: EphB1 Receptor Blockade Augments Morphine Antinociception via Inhibiting GRK2‐Mediated µ‐Opioid Receptor Internalization

doi: 10.1002/advs.78099

Figure Lengend Snippet: Activation of EphB1 receptors induces phosphorylation of MOR at Ser 375 and MOR internalization via GRK2. (A) Co‐IP analysis showing increased phosphorylation of GRK2 at tyrosine residues and enhanced interaction between GRK2 and MOR following treatment of ephrinB2‐Fc (0.01 µg/µL) in Neuro‐2a cells overexpressing MOR. The cell lysates were immunoprecipitated with an antibody against GRK2 and immunoblotted with PY99 antibodies to analyze the tyrosine phosphorylation of GRK2, or immunoblotted with Flag antibodies to analyze the interaction between GRK2 and MOR. Left, representative bands. Right, statistical summary. Each experiment is repeated three times. (B) Western blot showing that ephrinB2‐Fc‐induced phosphorylation of MOR at Ser 375 is inhibited by treatment of the GRK2 inhibitor, Paroxetine (5 µM). Upper, representative bands. Lower, statistical summary. Each experiment is repeated three times. (C) Immunofluorescence staining showing that Paroxetine (5 µM) inhibited ephrinB2‐Fc‐induced internalization of MOR in Neuro‐2a cells overexpressing MOR. Left, representative images. Scale bar indicates 10 µm. Right, statistical summary. n = 77 cells in IgG‐Fc + Vehicle group, n = 77 cells in IgG‐Fc + Paroxetine group, n = 65 cells in ephrinB2‐Fc + Vehicle group, and n = 90 in ephrinB2‐Fc + Paroxetine group. Statistical analysis by unpaired Student's t ‐test in (A) or one‐way ANOVA with Bonferroni's multiple comparison test in (B) and (C). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: DAMGO (HY‐P0210), DPDPE (HY‐P1334), Paroxetine (HY‐122272), and STA‐013 (HY‐179572) were purchased from MedChemExpress; U69593 (U103) was purchased from Sigma; Recombinant mouse ephrinB2‐Fc (496‐EB‐200) was purchased from R&D Systems; control mouse IgG‐Fc (JAC‐015‐000‐008) was purchased from Jackson ImmunoResearch.

Techniques: Activation Assay, Phospho-proteomics, Co-Immunoprecipitation Assay, Analysis, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Comparison