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.2014 Oct 10;289(41):28697-706.
doi: 10.1074/jbc.M114.588558. Epub 2014 Sep 2.

Heterologous regulation of Mu-opioid (MOP) receptor mobility in the membrane of SH-SY5Y cells

Affiliations

Heterologous regulation of Mu-opioid (MOP) receptor mobility in the membrane of SH-SY5Y cells

Kévin Carayon et al. J Biol Chem..

Abstract

The dynamic organization of G protein-coupled receptors in the plasma membrane is suspected of playing a role in their function. The regulation of the diffusion mode of the mu-opioid (MOP) receptor was previously shown to be agonist-specific. Here we investigate the regulation of MOP receptor diffusion by heterologous activation of other G protein-coupled receptors and characterize the dynamic properties of the MOP receptor within the heterodimer MOP/neuropeptide FF (NPFF2) receptor. The data show that the dynamics and signaling of the MOP receptor in SH-SY5Y cells are modified by the activation of α2-adrenergic and NPFF2 receptors, but not by the activation of receptors not described to interact with the opioid receptor. By combining, for the first time, fluorescence recovery after photobleaching at variable radius experiments with bimolecular fluorescence complementation, we show that the MOP/NPFF2 heterodimer adopts a specific diffusion behavior that corresponds to a mix of the dynamic properties of both MOP and NPFF2 receptors. Altogether, the data suggest that heterologous regulation is accompanied by a specific organization of receptors in the membrane.

Keywords: Bimolecular Fluorescence Complementation (BiFC); Fluorescence Recovery after Photobleaching (FRAP); G Protein-coupled Receptor (GPCR); Opiate Opioid; Plasma Membrane; Protein-Protein Interaction.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.
FIGURE 1.
Representative FRAP experiment performed at the observation radiusr = 3.17 μm in (SH2-D9)MOP-YFP cells.Gray squares represent measurements of the intensity of fluorescence before bleach and the intensity of fluorescence recovery over time after bleach. The fit (black line) of the data using a single population diffusion equation gives the mobile fractionM and τD diffusion time.
FIGURE 2.
FIGURE 2.
A, inhibition of potassium-evoked calcium influx by 30 s of treatment with 0. 1 μm DAMGO, 1 μm 1DMe, 10 μm clonidine, or 1 μm NPY. Thebars represent means ± S.E. of 48 (DAMGO), 15 (1DMe), 14 (clonidine), and 19 (NPY) cells.B, effect of 30 s of treatment with 0.1 μm DAMGO (control cells) on potassium-evoked calcium influx, in cells pretreated with 1 μm 1DMe, 10 μm clonidine, or 1 μm NPY for 30 min at room temperature. Thebars represent means ± S.E. of 48 (control), 47 (1DMe), 50 (clonidine), and 36 (NPY) cells. Intracellular calcium was measured using Fluo-4 AM. ***,p < 0.001, **,p < 0.01; different from control (one-way ANOVA followed by Dunnett's multiple comparison test).
FIGURE 3.
FIGURE 3.
Analysis of the lateral mobility of MOP-YFP receptors by FRAP experiments performed at the observation radiusr = 3.17 μm in (SH2-D9)MOP-YFP cells.A andB, effect of 1 μm 1DMe, 10 μm clonidine, and 1 μm NPY on the diffusion parametersM (A) andDapp (B) of the MOP-YFP receptor. FRAP experiments were conducted for 30 min at 20–22 °C in the absence (control) or in the presence of the different agonists. For each condition, recovery curves from 15–20 cells were recorded per coverslip, and the deducedM andDapp values were averaged. Thebars represent the means ± S.E. of theM andDapp values of 14–32 coverslips from several independent experiments. ***,p < 0.001, **,p < 0.01; different from control (one-way ANOVA followed by Dunnett's multiple comparison test).C andD, effect of 1 μm 1DMe or 10 μm clonidine on the diffusion parametersM (C) andDapp (D) of dC12-PE-NBD. The lipid (200 μm) was incubated for 10 min at room temperature. After a wash with 2 ml of PBS, FRAP measurement were performed in the absence (control) or in the presence of the agonists. Thebars represent the means ± S.E. of theM andDapp values of four to six coverslips from three independent experiments.
FIGURE 4.
FIGURE 4.
Internalization of the MOP-YFP receptor, visualized by confocal microscopy. SH2-D9(MOP-YFP) cells were incubated for 30 min at room temperature in HEPES-buffered medium, in the absence (control) or the presence of 1 μm DAMGO, 1 μm 1DMe, or 10 μm clonidine. Fixed cells were observed at λex 514 nm with a 60×/NA1.4 oil immersion objective on a confocal microscope (Olympus FV1000).
FIGURE 5.
FIGURE 5.
Analysis of the lateral mobility of MOP-YFP receptors by FRAP experiments performed at the observation radiusr = 3.17 μm in (SH2-D9)MOP-YFP cells.A, effect of overnight pretreatment with 100 ng/ml PTX on the regulation of the lateral mobility of MOP-YFP receptors by 1 μm 1DMe or 10 μm clonidine. FRAP measurement were performed for 30 min at 20–22 °C in cells not treated with PTX (control) and in PTX-treated cells in the absence (no agonist) or in the presence of agonists. Thebars represent the means ± S.E. of theM values of four to five coverslips from three experiments. *,p < 0.05; different from no agonist in PTX-treated cells (one-way ANOVA followed by Dunnett's multiple comparison test).B, effect of the opioid antagonist naloxone (1 μm) on the regulation of the lateral mobility of MOP-YFP receptors by 1 μm 1DMe or 10 μm clonidine. FRAP measurements were performed for 30 min at 20–22 °C in the absence (control) or in the presence of naloxone or in the presence of naloxone with each agonist. Thebars represent the means ± S.E. of theM values of nine coverslips from four experiments.
FIGURE 6.
FIGURE 6.
Characterization of the MOP/NPFF2 BiFC receptor.A, fluorescence complementation 48 h after transient transfection of HEK293 cells with various combinations of transmembrane receptors fused to Venus fragments (MOPVn/NPFF2Vc, CD8Vn/MOPVc, or CD8Vn/NPFF2Vc). Cell density was similar in each condition. Fixed cells were observed at λex 514 nm with a 40× oil immersion objective on a confocal microscope (Leica TCS SP5).B, confocal image of the stable SH-SY5Y cell line expressing the MOP/NPFF2 BiFC receptor showing expression at the plasma membrane.C, dose-response curve of the inhibition of forskolin-induced intracellular cAMP accumulation by 1DMe and DAMGO in SH-SY5Y cells expressing the MOP/NPFF2 BiFC receptor.Points represent means ± S.E. of at least three experiments performed in duplicate.
FIGURE 7.
FIGURE 7.
Internalization of the MOP/NPFF2 BiFC receptor in SH-SY5Y cells, visualized by confocal microscopy. Cells expressing the MOP/NPFF2 BiFC receptor were incubated for 30 min at room temperature in HEPES-buffered medium, in the absence (control) or the presence of 1 μm 1DMe or 1 μm DAMGO. Fixed cells were observed at λex 514 nm with a 60×/NA1.4 oil immersion objective on a confocal microscope (Olympus FV1000).
FIGURE 8.
FIGURE 8.
Comparison of the lateral mobility and domain sizes of MOP-YFP, NPFF2-YFP, and MOP/NPFF2 BiFC receptors in the membrane of SH-SY5Y cells by FRAP experiments at variable radius. FRAP experiments were conducted for 30 min at 20–22 °C, by using a set of diaphragms allowing observation of bleached areas of radius 1.88, 2.16, 2.80, 3.17, and 3.92 μm. For each condition, recovery curves from 15–20 cells were recorded per coverslip, and the deducedM values were averaged.Points represent means ± S.E. of theM values from 7–14 coverslips, plotted as a function of the inverse of the illumination radiusR.
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