Curr

Curr. requires -arrestin2, whereas Akt activation depends upon G protein signaling. These findings reveal a point of KOR transmission bifurcation that can be observed in an endogenous neuronal setting and may prove to be an important indication when developing biased agonists at the KOR. 0.6 nm, was obtained from Sigma. Mice -Arrestin2 knock-out (arr2-KO) and wild-type (WT) littermates were used to assess [35S]GTPS coupling in mouse brain and for the culturing of main neurons (28). All experiments were performed with the approval of the Institutional Animal Care and Use Committee of The Scripps Research Institute. [35S]GTPS Coupling CHO-KOR cells were collected, and membrane pellets were prepared by homogenization with a polytronic Tissue-Tearor in buffer (10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm EDTA) followed by 20,000 centrifugation at 4 C for 30 min. The resultant membrane pellet was resuspended in assay buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 5 mm MgCl2, 1 mm EDTA, 40 m GDP) via Teflon-on-glass Dounce homogenizer. Fifteen g of membrane protein was incubated with increasing concentrations of drug and 0.1 nm [35S]GTPS (PerkinElmer Life Sciences) for 1 h at 30 C in a total reaction volume of 200 l. For antagonist experiments, protein was preincubated with Rabbit Polyclonal to HDAC5 (phospho-Ser259) test compounds for 15 min prior to the addition of 100 nm U69,593 and [35S]GTPS. Reactions were terminated by quick filtration using a 96-well plate Brandel cell harvester (Brandel, Gaithersburg, MD) followed by washes with ice cold water. Microscint-20 (PerkinElmer Life Sciences) was added to the plates after drying, and radioactivity was read with a TopCount NXT HTS microplate scintillation and luminescence counter (PerkinElmer Life Sciences). All compounds were run in parallel assays in duplicate for comparison. For coupling in mouse brain, striata were isolated from 4C5-month-old male WT mice. Tissue was homogenized, and membranes were prepared as explained above. Coupling reactions, made up of 2.5 g of protein, 20 m GDP, and 0.1 nm [35S]GTPS, were incubated at room temperature for 2 h prior to harvesting, as explained above. -Arrestin2 Translocation To visually assess -arrestin2 translocation to the KOR, CHO-KOR cells were transiently transfected with arr2-GFP (5 g) by electroporation Bedaquiline fumarate and plated on collagen-coated glass-bottom confocal dishes (MatTek, Ashland, MA). After incubation at 37 C for 24 h, the cells were serum-starved for 1 h in serum-free MEM without phenol reddish (Invitrogen). Drugs were added, and live cell images were obtained at the time points indicated using an Olympus FluoView IX81 confocal microscope (Olympus, Center Valley, PA) as explained previously (29). -Arrestin2 translocation was also analyzed using the PathHunter? -arrestin assay in CHO-K1 cells expressing the KOR (DiscoveRx, Fremont, CA) according to the manufacturer’s protocol. Cells were treated with agonist for 90 min prior to assessment of enzyme complementation. For antagonist experiments, the cells were incubated with the antagonist for 60 min prior to agonist addition. Luminescence values were obtained using a Synergy HT luminometer (BioTek, Bedaquiline fumarate Winooski, VT). All compounds were run in parallel experiments in duplicate. KOR Internalization To qualitatively determine agonist-induced receptor internalization, CHO-KOR cells were plated on collagen-coated glass-bottom confocal dishes and serum-starved for 30 min prior to the experiment. The KOR Bedaquiline fumarate was labeled in live cells by incubation with an anti-HA-AlexaFluor 488 antibody (1:100, Invitrogen) for 15 min at 37 C. Following washes, drug (10 m) was added, and the cells were monitored by confocal imaging for HA-KOR internalization over the times indicated (29, 30). Agonist-induced KOR internalization was also assessed in CHO-KOR cells using a cell surface biotinylation assay. This assay was performed as explained previously (30). The cells were serum-starved for 30 min, and surface receptors were biotinylated prior to drug treatment. Drug was added at 10 m, and cells were treated for 30 min or 1 h (30 min for the antagonist and then 30 min for the agonist, where relevant). The remaining surface biotinylation was removed by glutathione-stripping buffer, and the guarded biotinylation was immunoprecipitated by streptavidin-coated beads. Western blots were performed as explained previously with 10% Bedaquiline fumarate bis-Tris NuPage gels (Invitrogen) and transferred to nitrocellulose membranes (18, 30). The receptor was detected using a KOR antibody (Neuromics, Edina, MN). Blots were imaged using the Odyssey infrared imager, and densitometry of bands was measured using Odyssey 2.1 software (Li-Cor, Lincoln, NE). The -fold activation over vehicle was calculated by dividing the densitometry values of the treated groups by the average value of the vehicle-treated group. In-cell Western To assess agonist-induced ERK1/2 phosphorylation via In-cell.