Journal: Biochimica et biophysica acta

Article Title: Heterodimerization of the kappa opioid receptor and neurotensin receptor 1 contributes to a novel β-arrestin-2-biased pathway.

doi: 10.1016/j.bbamcr.2016.07.009

Figure Lengend Snippet: Fig. 1. Co-localization of KOR and NTSR1 in the primary culturedstriatal neurons. (A) Anti-KOR and anti-NTSR1 antibodies detected 43 kDa (upper panels) and 46 kDa (lower panels) protein bands, respectively, in western blot analysis of lysates of the primary cultured rat striatal neurons. (B) Striatal neurons stained with antibodies against KOR and NTSR1 were visualized using laser confocal microscopy. Cell nuclei were stained with DAPI, a DNA-specific dye (blue). Confocal images of KOR (red) and NTSR1 (green) were merged to reveal regions of co-localization (yellow). (C) In situ PLA analysis was performed in cultured striatal neurons to detect the interaction between endogenously expressed KOR and NTSR1. Anti- KOR and anti-NTSR1 antibodies were used. Punctate fluorescent signals (a, red dots), indicating a close proximity of KOR and NTSR1 leading to heterodimer formation, were revealed by confocal microscopy. Neurons incubated without anti-KOR and anti-NTSR1 antibodies were used as negative controls (b).

Article Snippet: After rinsing three times with TBST, the blots were incubated with rabbit anti-phospho-ERK1/2 antibody (Cell Signaling Technology) (1:2000) overnight at 4 °C, to assess the phosphorylation status of ERK1/2 in KOR and/or NTSR1 stably expressing HEK293 cells; blots were incubated with mouse anti-Myc or rabbit anti-HA antibodies (Cell Signaling Technology) (1:2000), to detect the expression status ofWT ormutated KORs or NTSR1 in stably transfected HEK293 cells; or blots were incubated with goat anti-KOR antibody (Novus Biologicals) (1:500) and rabbit anti-NTSR1 antibody (Abcam) (1:500), to detect the expression of KOR or NTSR1 in the primary cultured neurons.

Techniques: Western Blot, Cell Culture, Staining, Confocal Microscopy, In Situ, Incubation

Journal: Biochimica et biophysica acta

Article Title: Heterodimerization of the kappa opioid receptor and neurotensin receptor 1 contributes to a novel β-arrestin-2-biased pathway.

doi: 10.1016/j.bbamcr.2016.07.009

Figure Lengend Snippet: Fig. 2. Co-expression and co-localization of KORWT (KOR184 or KOR196) and NTSR1 in HEK293 cells. (A) Analysis of the co-localization of NTSR1 and KORWT (KOR184 or KOR196) in HEK293 cells. HA–NTSR1 (green) and Myc–KORWT (Myc–KOR184or Myc–KOR196; red) were stably expressed in HEK293 cells and visualized using laser confocal microscopy. Cell nuclei were stained with DAPI (blue). Confocal images of NTSR1 and KOR were merged to reveal regions of co-localization (yellow). (B) Expression levels of Myc–KOR or HA–NTSR1 proteins in HEK293 cells stably expressing Myc–KOR or HA–NTSR1, or HEK293 cells stably co-expressing Myc–KOR and HA–NTSR1, were determined by western blotting. (C) Determination of cell surface expression of Myc–KORs and HA–NTSR1. ELISA was conducted with intact cells using anti-Myc and anti-HA antibodies. Myc–KOR and HA–NTSR1 were used to transfect HEK293 cells, singly or together, as illustrated in the figure. Values are means ± SEM of triplicate determinations (with at least ten samples per group in each independent experiment). Differences in expression were analyzed by one-way ANOVA followed by Tukey's multiple comparison test (ns, not significant, P N 0.05).

Article Snippet: After rinsing three times with TBST, the blots were incubated with rabbit anti-phospho-ERK1/2 antibody (Cell Signaling Technology) (1:2000) overnight at 4 °C, to assess the phosphorylation status of ERK1/2 in KOR and/or NTSR1 stably expressing HEK293 cells; blots were incubated with mouse anti-Myc or rabbit anti-HA antibodies (Cell Signaling Technology) (1:2000), to detect the expression status ofWT ormutated KORs or NTSR1 in stably transfected HEK293 cells; or blots were incubated with goat anti-KOR antibody (Novus Biologicals) (1:500) and rabbit anti-NTSR1 antibody (Abcam) (1:500), to detect the expression of KOR or NTSR1 in the primary cultured neurons.

Techniques: Expressing, Stable Transfection, Confocal Microscopy, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Biochimica et biophysica acta

Article Title: Heterodimerization of the kappa opioid receptor and neurotensin receptor 1 contributes to a novel β-arrestin-2-biased pathway.

doi: 10.1016/j.bbamcr.2016.07.009

Figure Lengend Snippet: Fig. 3. FRET and BRET analyses of heterodimerization of KOR (WT or mutated) and NTSR1. (A) FRET imaging of constitutive KOR–NTSR1 heterodimeric interactions in living cells. HEK293 cells were transiently transfected (protein co-expression or individual expression) with the appropriate plasmids, as follows: (a) NTSR1–eCFP, (b) KORWT–Venus, (c) NTSR1–eCFP/KORWT– Venus, (d) KOR184–Venus, (e) NTSR1–eCFP/KOR184–Venus, (f) KOR196–Venus, (g) NTSR1–eCFP/KOR196–Venus. Empty pcDNA3.1 vector was used to equilibrate the total amount of transfected cDNA. Left-hand panels, donor; center panels, acceptor; right-hand panels, corrected FRET signal. (B) Calculated normalized FRET values, assessed as described in Experimental Procedures. ⁎⁎P b 0.001, NTSR1–eCFP/KOR196–Venus group vs. NTSR1–eCFP/KORWT–Venus group (or NTSR1–eCFP/KOR184–Venus group); ns, not significant, P N 0.05. Results are means ± SEM of at least three independent experiments (with ten samples per group in each independent experiment). (C) HEK293 cells were transiently transfected with increasing ratios of KORWT–Venus (KOR184–Venus, KOR196–Venus, or Venus) to NTSR1–Rluc, and various amounts of empty pcDNA3.1 plasmid DNA were added to maintain a constant amount of total transfected plasmid DNA. Total luminescence and Venus fluorescence were measured at the beginning of each experiment to obtain the intensity ratio of fluorescence/ total luminescence, and the BRET ratio was calculated as Emission 535/Emission 460. NTSR1–Rluc and KOR184–Venus (green dots), as well as NTSR1–Rluc and KORWT–Venus (red dots), displayed a significant specific interaction, while NTSR1–Rluc and KOR196–Venus (blue dots), and NTSR1–Rluc and Venus (negative control, black dots), showed non-specific interaction. (D) BRET signals were analyzed as indicated. Rluc and Venus fusion constructs were used to transfect HEK293 cells at a 1:3 protein ratio. Energy-transfer measurements were performed in living cells by adding coelenterazine and measuring light emission in a luminometer with Rluc and Venus filter settings, as described above. ⁎⁎P b 0.001 vs. the NTSR1–Rluc/Venus group; ns, not significant; P N 0.05. Data are representative of at least three independent experiments shown in Fig. 3.

Article Snippet: After rinsing three times with TBST, the blots were incubated with rabbit anti-phospho-ERK1/2 antibody (Cell Signaling Technology) (1:2000) overnight at 4 °C, to assess the phosphorylation status of ERK1/2 in KOR and/or NTSR1 stably expressing HEK293 cells; blots were incubated with mouse anti-Myc or rabbit anti-HA antibodies (Cell Signaling Technology) (1:2000), to detect the expression status ofWT ormutated KORs or NTSR1 in stably transfected HEK293 cells; or blots were incubated with goat anti-KOR antibody (Novus Biologicals) (1:500) and rabbit anti-NTSR1 antibody (Abcam) (1:500), to detect the expression of KOR or NTSR1 in the primary cultured neurons.

Techniques: Imaging, Transfection, Expressing, Plasmid Preparation, Negative Control, Construct

Journal: Biochimica et biophysica acta

Article Title: Heterodimerization of the kappa opioid receptor and neurotensin receptor 1 contributes to a novel β-arrestin-2-biased pathway.

doi: 10.1016/j.bbamcr.2016.07.009

Figure Lengend Snippet: Fig. 4. KOR-L196 A mutation impairs KOR interaction with NTSR1, as assessed by CO-IP assays. (A) HEK293 cells were transfected with the expression vectors for both HA–NTSR1 and Myc– KORWT (“Co-transfected N-KWT”) or with either vector alone; or with the expression vectors for both HA–NTSR1 and Myc–KOR184 (“Co-transfected N-K184”) or with either vector alone. Samples containing either HA–NTSR1 or Myc–KORWT were mixed (“Mix N-KWT”); samples containing either HA–NTSR1 or Myc–KOR184 were also mixed (“Mix N-K184”). (B) HEK293 cells were transfected with the expression vectors for both HA–NTSR1 and Myc–KORWT (“Co-transfected N-KWT”) or with either vector alone; or with the expression vectors for both HA– NTSR1 and Myc–KOR196 (“Co-transfected N-K196”) or with either vector alone. Samples containing either HA–NTSR1 or Myc–KORWT were mixed (“Mix N-KWT”), and samples containing either HA–NTSR1 or Myc–KOR196 were also mixed (“Mix N-K196”). Cell lysates were subsequently immunoprecipitated (IP) with anti-HA agarose beads and immunoblotted with anti- Myc antibodies (upper panels). Verification of the expression of appropriate constructs was obtained after immunoblotting the cell lysates with either anti-HA or anti-Myc antibodies (lower panels), as required.

Article Snippet: After rinsing three times with TBST, the blots were incubated with rabbit anti-phospho-ERK1/2 antibody (Cell Signaling Technology) (1:2000) overnight at 4 °C, to assess the phosphorylation status of ERK1/2 in KOR and/or NTSR1 stably expressing HEK293 cells; blots were incubated with mouse anti-Myc or rabbit anti-HA antibodies (Cell Signaling Technology) (1:2000), to detect the expression status ofWT ormutated KORs or NTSR1 in stably transfected HEK293 cells; or blots were incubated with goat anti-KOR antibody (Novus Biologicals) (1:500) and rabbit anti-NTSR1 antibody (Abcam) (1:500), to detect the expression of KOR or NTSR1 in the primary cultured neurons.

Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Construct, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Heterodimerization of the kappa opioid receptor and neurotensin receptor 1 contributes to a novel β-arrestin-2-biased pathway.

doi: 10.1016/j.bbamcr.2016.07.009

Figure Lengend Snippet: Fig. 5. Profiling the KOR/NTSR1 interaction–mediated G protein signaling. (A–C) The KOR–NTSR1 interaction attenuated dynorphin A(1–13)–induced inhibition of cAMP accumulation in HEK293-KORWT/NTSR cells. HEK293 cells stably expressing KORWT and/or NTSR1 (A), KOR184 and/or NTSR1 (B), or KOR196 and/or NTSR1 (C) were pre-incubated with IBMX (0.5 mM), followed by 15 min of incubation with 10 μM forskolin in the absence or presence of different doses of dynorphin A(1–13) (0.01–10,000 nM) and/or NT(8–13) (0.001–1000 nM), as indicated [DYN: dynorphin A(1–13); NT: NT(8–13)]. Absorbance-based cAMP ELISA kits were used to detect intracellular cAMP levels. The data correspond to three independent experiments carried out in triplicate, and changes inintracellular cAMP levels were quantified and expressed as % forskolin response. (D–F) Profiling the KOR/NTSR1 interaction– mediated activation of the Gαi2 protein subunit using BRET analysis. Real-time analysis of the interaction between Rluc–Gαi2 and KORWT–Venus, with or without untagged NTSR1(D); Rluc–Gαi2 and KOR184–Venus, with or without untagged NTSR1 (E); and Rluc–Gαi2 and Venus–KOR196, with or without untagged NTSR1 (F), employing eBRET, in living cells. After the cells were incubated with EnduRen™for 2 h, BRET signals were recorded following stimulation by agonists, 100 nM DYN and/or 10 nM NT (⁎P b 0.05, DYN-treated HEK293 cells co- expressing KORWT/184 and NTSR1 vs. DYN-treated HEK293 cells expressing only KORWT/184; ⁎⁎P b 0.05, DYN-treated HEK293 cells expressing only KORWT/184/196 vs. HEK293 cells expressing only KORWT/184/196 treated with vehicle; ns, P N 0.05, DYN + NT–treated HEK293 cells co-expressing KORWT/184/196 and NTSR1 vs. DYN-treated HEK293 cells expressing only KORWT/184/196; Student's t-test). The data represent means ± SEM from three independent experiments (with at least ten samples per group in each independent experiment).

Article Snippet: After rinsing three times with TBST, the blots were incubated with rabbit anti-phospho-ERK1/2 antibody (Cell Signaling Technology) (1:2000) overnight at 4 °C, to assess the phosphorylation status of ERK1/2 in KOR and/or NTSR1 stably expressing HEK293 cells; blots were incubated with mouse anti-Myc or rabbit anti-HA antibodies (Cell Signaling Technology) (1:2000), to detect the expression status ofWT ormutated KORs or NTSR1 in stably transfected HEK293 cells; or blots were incubated with goat anti-KOR antibody (Novus Biologicals) (1:500) and rabbit anti-NTSR1 antibody (Abcam) (1:500), to detect the expression of KOR or NTSR1 in the primary cultured neurons.

Techniques: Inhibition, Stable Transfection, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Biochimica et biophysica acta

Article Title: Heterodimerization of the kappa opioid receptor and neurotensin receptor 1 contributes to a novel β-arrestin-2-biased pathway.

doi: 10.1016/j.bbamcr.2016.07.009

Figure Lengend Snippet: Fig. 9. BRET and PLA profiling of the KOR–NTSR1 interaction. (A) The dose-response curve of the interaction between KOR and NTSR1 was measured by BRET1. HEK293 cells transiently transfected with Rluc–NTSR1 and Venus–KORWT (KOR–Venus) were treated with increasing doses of dynorphin A(1–13) (DYN) or NT(8–13) (NT), or increasing NT at a fixed final DYN concentration (100 nM). BRET signals between NTSR1–Rluc and KOR–Venus were recorded 30 min after agonist administration. The results represent means ± SEM from three independent experiments (with at least ten samples per group in each independent experiment). (B) Kinetic response analysis of the interaction between KOR and NTSR1 using eBRET in living cells. NTSR1–Rluc and KOR–Venus constructs were used to transfect HEK293 cells at a 1:3 protein ratio. The cells were then incubated with EnduRen™for 2 h, and BRET signals were recorded following stimulation with agonists (100 nM DYN or/and 10 nM NT). The data are presented as means ± SEM from three independent experiments (with at least ten samples per group in each independent experiment). (C) The specificity of in situ PLA was determined in HEK293 cells stably expressing KORWT and/or NTSR1, with or without 30 min of pre-incubation with ligands (100 nM DYN, 10 nM NT), as indicated. Thereafter, the punctate fluorescent signal suggesting close proximity of KOR and NTSR1 leading to heterodimer formation was detected by confocal microscopy (c–f) (c: vehicle; d: DYN-treated; e: NT-treated; f: DYN + NT–treated), with HEK293 cells only expressing either KOR or NTSR1 as negative controls (a or b). (D) Specificity of the in situ PLA signals was quantified as blobs/cell signifying the KOR–NTSR1 interaction. $$P b 0.001, vs. control group; &&P b 0.001, DYN-pretreated group vs. NT- or NT/DYN-pretreated group; ns, P N 0.05, not statistically significant. The data represent means ± SEM of three measurements from three independent experiments.

Article Snippet: After rinsing three times with TBST, the blots were incubated with rabbit anti-phospho-ERK1/2 antibody (Cell Signaling Technology) (1:2000) overnight at 4 °C, to assess the phosphorylation status of ERK1/2 in KOR and/or NTSR1 stably expressing HEK293 cells; blots were incubated with mouse anti-Myc or rabbit anti-HA antibodies (Cell Signaling Technology) (1:2000), to detect the expression status ofWT ormutated KORs or NTSR1 in stably transfected HEK293 cells; or blots were incubated with goat anti-KOR antibody (Novus Biologicals) (1:500) and rabbit anti-NTSR1 antibody (Abcam) (1:500), to detect the expression of KOR or NTSR1 in the primary cultured neurons.

Techniques: Transfection, Concentration Assay, Construct, Incubation, In Situ, Stable Transfection, Expressing, Confocal Microscopy, Control