PMC

A: A dendrite co-expressing immunoperoxidase labeled D1R (arrows) and immunogold labeled DOR (arrowheads) in the NAcbS. D1R is visualized along the plasma membrane while DOR is located intracellularly. B: Reverse labeling illustrating a dendrite in the NAcbC exhibiting co-localization of immunoperoxidase labeled DOR (arrows) and immunogold labeled D1R (arrowheads). An unlabeled axon terminal containing vesicles is apposed to the co-labeled dendrite. Bar = 500nm. Abbreviations: D1R + DOR-d = D1R and DOR labeled dendrite; ut = unlabeled terminal; D1R-d = D1R labeled dendrite; ma= myelinated axon: m=mitochondria. C: Bar graphs representing the percentage of co-localization between DOR and D1R. In the NAcbS 33% of D1R labeled profiles contained DOR immunolabeling and 34% of DOR labeled profiles contained D1R immunoreactivity. In the NAcbC, 24% of D1R labeled profiles contained DOR immunoreactivity and 38% of DOR labeled profiles contained immunolabeling for D1R.

The C-terminal tail of DOR regulates delivery of the receptor to the cell surface. (A) The last 27 amino acids of the DOR C-terminal tail were subdivided into four deletion mutants. In a Flag-tagged DOR construct, deletion mutants were made to determine the region within the DOR C-terminal tail required for the NGF-induced Golgi retention of DOR (Δ345–352, Δ353–359, Δ360–366, and Δ367 TGA). In PC12 cells expressing the wild-type Flag-tagged DOR (DOR WT), immunofluorescence images reveal that DOR is highly expressed on the cell surface under control no-treatment (NT) conditions. DOR accumulates in a region that colocalizes with the trans-Golgi network marker TGN-38 (yellow arrow) following 1 h of nerve growth factor treatment (NGF, 100 ng/ml). Sectional deletions of the regions comprising the 27 amino acids of the C-terminal DOR tail sequence all demonstrate baseline surface localization under control NT conditions, and accumulation of DOR within the Golgi region following NGF treatment. (B) Quantitative analysis of the immunofluorescence images showed an increase in the percentage of DOR fluorescence localized to the Golgi region following NGF treatment for cells expressing DOR WT. Of the individual DOR tail-deletion mutants, the Δ345–352 and the Δ353–359 both displayed a small reduction in the percentage of DOR fluorescence localized to the Golgi following NGF treatment, with only Δ353–359 showing a significant reduction compared with DOR WT NGF. The remaining deletion mutants were similar to DOR WT under both NT and NGF treatment conditions (DOR WT NT, n = 82 cells; DOR WT NGF, n = 77 cells; Δ345–352 NT, n = 97 cells; Δ345–352 NGF, n = 98 cells; Δ353–359 NT, n = 77 cells; Δ353–359 NGF, n = 105 cells; Δ360–366 NT, n = 84 cells; Δ360–366 NGF, n = 80 cells; Δ367 TGA NT, n = 34 cells; Δ367 TGA NGF, n = 39 cells; mean ± SEM; *p < 0.05, by one-way ANOVA with Dunnett’s multiple comparisons test vs. DOR WT NGF). (C). Additional quantification of the percentage of cells that appeared to have Golgi-localized DOR demonstrated a pattern similar to the fluorescence quantification. The Δ345–352 and the Δ353–359 DOR tail deletions displayed a small, but significant reduction in the percentage of cells with Golgi-localized DOR following NGF treatment compared with DOR WT NGF (DOR WT NT, n = 82 cells; DOR WT NGF, n = 77 cells; Δ345–352 NT, n = 97 cells; Δ345–352 NGF, n = 98 cells; Δ353–359 NT, n = 77 cells; Δ353–359 NGF, n = 105 cells; Δ360–366 NT, n = 84 cells; Δ360–366 NGF, n = 80 cells; Δ367 TGA NT, n = 34 cells; Δ367 TGA NGF, n = 39 cells; mean ± SEM; **p < 0.01, ****p < 0.0001; by a two-tailed chi-squared test vs. DOR WT NGF).

Diarginine motifs in the third intracellular loop and C-terminal tail of DOR are not required for NGF-induced DOR retention. (A) Alanine mutant versions of a Flag-tagged DOR were made containing SLRRPR to AAAAAA (a) and RSLRR to AAAAA (b). (B) Immunofluorescence imaging of PC12 cells expressing a wild-type Flag-tagged DOR (DOR WT) or alanine mutated diarginine containing motifs demonstrated primarily surface localization under normal baseline no-treatment conditions (NT). Following NGF treatment for 1 h (NGF; 100 ng/ml) or PI3K inhibition by LY294002 (LY; 10 µM), all DOR constructs exhibited increased Golgi localization. (C) Quantification of the percentage of DOR fluorescence localized to the Golgi was normalized to DOR WT with NT to compare the alanine-mutated diarginine-containing motifs with the full-length DOR. Mutating the SLRRPRΔAla and RSLRRΔAla did not significantly affect the NGF-induced DOR-Golgi localization. Mutating the SLRRPRΔAla and RSLRRΔAla alone resulted in a significant decrease in the DOR fluorescence within the Golgi following PI3K inhibition by LY compared with DOR WT LY (DOR WT NT, n = 58 cells; DOR WT NGF, n = 56 cells; DOR WT LY, n = 67 cells; SLRRPRΔAla NT, n = 49 cells; SLRRPRΔAla NGF, n = 91 cells; SLRRPRΔAla LY, n = 85 cells; RSLRRΔAla NT, n = 40 cells; RSLRRΔAla NGF, n = 52 cells; RSLRRΔAla LY, n = 91 cells; mean ± SEM; ***p < 0.001, ****p < 0.0001, by one-way ANOVA with Dunnett’s multiple comparison test vs. DOR WT, NGF and LY). (D) Analysis of the percentage of cells with Golgi-localized DOR showed a small but significant decrease between the DOR WT LY and the SLRRPRΔAla and RSLRRΔAla alone (DOR WT NT, n = 58 cells; DOR WT NGF, n = 56 cells; DOR WT LY, n = 67 cells; SLRRPRΔAla NT, n = 49 cells; SLRRPRΔAla NGF, n = 91 cells; SLRRPRΔAla LY, n = 85 cells; RSLRRΔAla NT, n = 40 cells; RSLRRΔAla NGF, n = 52 cells; RSLRRΔAla LY, n = 91 cells; mean ± SEM; **p < 0.01, by two-tailed chi-squared test vs. DOR WT, NGF and LY).

A critical role of δ-opioid receptor (DOR) for anti-microglial activation. NC, negative control; DOR KD, DOR knockdown; DOR OV, DOR overexpression. (A) RT-PCR evaluation of the efficiency of plasmids infection. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. NC. Note that the DOR knockdown shRNA plasmids infections significantly reduced DOR mRNA expression in BV2 cells, especially the 103885-1 and 103887-2. The BV2 cells infected with 71788-1 plasmids showed folds increases in DOR mRNA compared to the negative control. (B) DOR knockdown or overexpression-induced alterations in MHCII and arginase protein expression. **p < 0.01, ***p < 0.001 vs. NC. Note that DOR knockdown caused an upregulation of both MHCII and arginase, while DOR overexpression did not seriously affect MHCII and arginase expression. (C) Effects of DOR knockdown or overexpression on MOR and KOR of BV2 cells. **p < 0.01, ***p < 0.001 vs. NC. Note that 103885-1 and 103887-2, the DOR knockdown shRNA plasmids significantly reduced DOR mRNA expression in BV2 cells. The BV2 cells infected with 71788-1 plasmids showed folds increases in DOR mRNA compared to the negative control. Neither DOR knockdown nor overexpression caused any appreciable change in MOR and DOR expression by RT-PCR or Western blot measurements.