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, 281 (26), 18081-9

Kappa Opioid Receptor Activation of p38 MAPK Is GRK3- And Arrestin-Dependent in Neurons and Astrocytes

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Kappa Opioid Receptor Activation of p38 MAPK Is GRK3- And Arrestin-Dependent in Neurons and Astrocytes

Michael R Bruchas et al. J Biol Chem.

Abstract

AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (gamma-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism.

Figures

FIGURE 1
FIGURE 1. KOR activation of p38 MAPK is phosphorylation- and arrestin-dependent in AtT-20 cells
KOR-GFP- and KSA-GFP-expressing AtT-20 cells were grown on cover-slips as described under “Experimental Procedures.” Cells were then fixed and labeled with anti-phospho-p38 MAPK (red), and green fluorescence is the GFP signal corresponding to the KOR or KSA receptor. Blue fluorescence corresponds to the Topro3 nuclear staining. KOR-GFP receptor was visualized (A) in the absence of U50,488 treatment. Following 10 μM U50,488 treatment for 15 min at 37 °C (B), KOR-GFP was internalized and phospho-p38 MAPK staining was increased. The inset in B shows KOR-GFP cells pretreated with 1 μM norBNI, prior to U50,488 treatment, and shows blockade of KOR-mediated phospho-p38 staining. KSA-GFP receptors distribute in a similar manner to the KOR in the absence of agonist (C ); however, they did not internalize or increase phospho-p38 MAPK upon the addition of 10 μM U50,488 for 15 min (D). Following transfection of the dominant positive arrestin R170E, the KSA-GFP receptor is able to respond to U50,488 treatment as demonstrated by its internalization (F ) and phospho-p38 MAPK (F, inset) activation; white arrows point to phospho-p38 KSA-GFP overlay. All experiments were performed on 2– 4 independent experiments.
FIGURE 2
FIGURE 2. Dominant positive arrestin restores KSA-GFP mediated p38 MAPK activation
KSA-GFP-expressing AtT-20 cells were transiently transfected with the dominant positive arrestin (R170E) and then exposed to U50,488 treatment. A, mean band intensities expressed as a percentage of basal-untreated control ± S.E. of KSA-GFP-expressing AtT-20 cells. Following 10 μM U50,488 treatment for 15 and 30 min (KSA U50 15, KSA U50 30), phospho-p38 levels were not significantly different from untreated controls. In KSA-GFP/R170E cells, 10 μM U50,488 treatment for 15 or 30 min (R170E U50 15, R170E U50 30) facilitated agonist-dependent p38 MAPK activation. n = 3, from separate transfections and experiments. *, significantly different from basal, p < 0.05, using the student’s t test. P-p38, phospho-p38 MAPK. B, representative Western blot data for phospho-p38 MAPK (P-p38) in KSA-GFP expressing A + T-20 cells.
FIGURE 3
FIGURE 3. KOR-GFP but not KSA-GFP mediates p38 phosphorylation following agonist treatment
A–C, AtT-20 cells expressing either KOR-GFP or KSA-GFP were treated with 10 μM U50,488 for different times at 37 °C and then resolved by Western blot. A, representative Western blot data for phospho-p38 MAPK (P-p38) and β-actin protein loading control in KOR-GFP-expressing AtT-20 cells. B, representative Western blot data for phospho-p38 MAPK and β-actin protein loading in KSA-GFP AtT-20 cells. C, ■ are the mean band intensities expressed as a percentage of basal-untreated control ± S.E. of KOR-GFP mediated p38 MAPK phosphorylation; □ represent mean band intensities expressed as a percentage of basal-untreated control ± S.E. from AtT-20 cells expressing KSA-GFP. Phospho-p38 levels were only significantly (*) increased at the 15-min post-U50,488 time point for wild-type KOR-GFP. n = 3– 4, where each n represents an independent experiment. *, significantly different from basal, p < 0.05 using the student’s t test. D, representative Western blot data for phospho-ERK 1/2 (P-ERK 1/2) and β-actin protein loading control in KOR-GFP-expressing AtT-20 cells. E, representative Western blot data for phospho-ERK 1/2 MAPK and β-actin protein loading in KSA-GFP AtT-20 cells. F, ■ are the mean band intensities expressed as a percentage of basal-untreated control ± S.E. of KOR-GFP mediated ERK 1/2 phosphorylation; □ represent mean band intensities expressed as a percentage of basal-untreated control ± S.E. from AtT-20 cells expressing KSA-GFP and show a similar increase in the relative U50,488-stimulated ERK 1/2 activation. n = 3– 4, where each n represents an independent experiment. *, significantly different from basal for both KOR-GFP and KSA-GFP groups, p < 0.05 using the student’s t test.
FIGURE 4
FIGURE 4. Primary cultures of striatal astrocytes show increased phospho-p38 MAPK (P-p38) activation in response to KOR stimulation
Primary cultures of striatal astrocytes were grown on coverslips as described under “Experimental Procedures.” Cultures were treated with either vehicle or 10 μM U50,488 for 15 min. A–C show vehicle (basal) phospho-p38 (A, fluorescein isothiocyanate, green) and GFAP (B, rhodamine, red) merged (C, yellow) and demonstrate minimal phospho-p38 staining in the absence of agonist. However, D–F show increased 10 μM U50,488-stimulated phospho-p38 (D, green). This increase in phospho-p38 is colocalized with the astrocyte marker, GFAP (E, red) colocalization (F, yellow). The white arrows point to cells with particularly bright overlay following U50,488 treatment. Images shown are representative of at least 4 separate experiments from individual primary cultures.
FIGURE 5
FIGURE 5. KOR-mediated p38 activation in AtT-20 cells and Striatal Astrocytes is concentration-dependent
A, phospho-p38 MAPK (P-p38) concentration-response curves for the KOR agonists U50,488 and the endogenous KOR agonist-peptide dynorphin B in AtT-20 cells expressing KOR-GFP. B, phospho-p38 concentration-response curves for the KOR agonists U50,488 and salvinorin A in primary striatal astrocytes. All agonist treatments were performed at the 15-min time point. n = 3– 4, with each n taken from a separate cell culture and experiment.
FIGURE 6
FIGURE 6. U50,488-stimulated p38-MAPK activation in primary striatal astrocytes and neurons is KOR-mediated and GRK3-dependent
A and B, wild type (W T), kappa opioid receptor knock-out (KOR−/−), and G-protein-coupled receptor kinase knock-out (GRK3−/−) primary striatal astrocytes were cultured as described under “Experimental Procedures.” Astrocyte cultures were treated with vehicle (B), 10 μM U50,488 (U50), or U50,488 in the presence of 1 μM norbinaltorphimine (NBNI, 1-h pretreatment) for 15 min at 37 °C. KOR activation resulted in phosphorylation of p38 MAPK (p-p38) that was blocked by NBNI. U50,488-stimulated p38 activation was not evident in astrocytes from KOR−/−mice. In addition, U50,488 treatment did not induce p38 activation in astrocytes from GRK3−/− mice. In KOR−/− or GRK3−/− mice, anisomycin (ANIS), an efficacious activator of p38 MAPK (50 μM, 15 min, 37 °C), effectively stimulated p38 severalfold over basal, demonstrating an intact p38 MAPK system in these cultures. A, representative Western blots for phospho-p38 from W T, KOR−/−, and GRK3−/−primary striatal astrocytes. B, representative Western blots for phospho-p38 from W T, KO−/−, and GRK3−/−primary striatal neurons. C, data are the mean ± S.E. of U50,488-stimulated phospho-p38 in astrocytes from W T, KOR−/−, and GRK3−/−mice as a percentage of basal (dashed line). n = 7– 8, with each n taken from separate primary cell cultures and experiments. *, p < 0.05 for W T astrocytes plus U50,488 versus basal using the student’s t test. C and D, W T kappa opioid receptor knock-out (KOR−/−) and G-protein-coupled receptor kinase knock-out (GRK3−/−) primary striatal neurons were grown in culture as described under “Experimental Procedures.” KOR activation resulted in phosphorylation of p38 MAPK (p-p38) that was blocked by NBNI. U50,488-stimulated p38 activation was not evident in neurons from either KOR−/− mice or GRK3−/− mice. D, data are the mean ± S.E. of U50,488-stimulated phospho-p38 in neurons from W T, KOR−/−, and GRK3−/− mice as a percentage of basal (dashed line). n = 8 –10, with each n taken from separate primary cell cultures and experiments. **, p < 0.01 for W T astrocytes plus U50,488 versus basal using analysis of variance and Dunnett’s post hoc test.
FIGURE 7
FIGURE 7. Primary cultures of striatal neurons showincreasedphospho-p38MAPKco-localized with arrestin3 in response to KOR stimulation
Primary striatal neurons were grown on coverslips as described under “Experimental Procedures.” Cultures were treated with either vehicle (basal) or 10 μM U50,488 for 15 min. A–C, show vehicle (basal) phospho-p38 (A, Alexa Fluor 488, green) and arrestin3 (B, Alexa Fluor 555, red) merged (C ) and demonstrate diffuse phospho-p38 labeling in the absence of agonist. However, E–G show a clustered, punctate, and increased staining for 10 μM U50,488-stimulated phospho-p38 (E) colocalized with arrestin3 (G, yellow). Images shown are representative of at least 4 separate experiments from individual primary cultures.
FIGURE 8
FIGURE 8. U50,488-stimulated phospho-p38 is decreased in arrestin3 siRNA-treated astrocytes
Wild-type primary striatal astrocytes were grown in culture as described. Individual astrocyte cultures were treated with siRNA for arrestin3 as outlined under “Experimental Procedures.” A, representative Western blot for the 55-kDa protein arrestin3 (Arr3) in primary striatal astrocytes in the presence and absence (+,−) of siRNA for arrestin3; the β-actin control for protein loading and similar protein levels following siRNA treatment is also shown. B, data are the mean ± S.E. of U50,488-mediated phospho-p38 in striatal astrocytes in the absence (Control (+U50)) and presence of siRNA for arrestin3 (siRNA (+U50)). n = 6, with each n taken from separate primary cultures and siRNA treatment experiments. *, p < 0.05 using the students t test.

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