Ross E

Ross E. both Gi and Go with a bias toward Go, but RGS7/G5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins. luciferase (GeneTex) were purchased. Recombinant His-tagged RGS7 and Pik3r2 RGS9-2 were coexpressed with G5S in Sf9 insect cells, and the complexes were purified as described previously (24). Cell Culture and Transfection HEK293T/17 cells were grown in DMEM supplemented with 10% FBS, minimum Eagle’s medium non-essential amino acids, 1 mm sodium pyruvate, and antibiotics (100 units/ml penicillin and 100 g/ml streptomycin) at 37 C in a humidified incubator containing 5% CO2. For transfection, cells were seeded into 6-cm dishes at a density of 4 106 cells/dish. After 4 h, expression constructs (total 5 g/dish) were transfected into the cells using PLUS (5 l/dish) and Lipofectamine LTX (8 l/dish) reagents. The GPCR (-opioid receptor or dopamine D2 receptor), G (Go, Gi1, Gz, Gq, or Gs), Venus 156-239-G, Venus 1C155-G2, masGRK3ct-Rluc8, G5S, and R7BP constructs were transfected at a 1:2:1:1:1:1:1 ratio with different amounts of R7 RGS (RGS7 or RGS9-2). An empty vector was used to normalize the amount of transfected DNA. Fast Kinetic BRET Assay Agonist-dependent cellular measurements of BRET between masGRK3ct-Rluc8 and G12-Venus were performed to visualize the action of G protein signaling in living cells, as described previously, with slight modifications (25). 16 to 24 h post-transfection, HEK293T/17 cells were washed once with PBS containing 5 mm EDTA (EDTA/PBS) and detached by incubation in EDTA/PBS at room temperature for 10 min. Cells were harvested with centrifugation at 500 g for 5 min and resuspended in PBS containing 0.5 mm MgCl2 and 0.1% glucose (BRET buffer). Approximately 50,000C100,000 cells/well were distributed in 96-well flat-bottomed white microplates (Greiner Bio-One). The Rluc substrate, coelenterazine-(Nanolight Technologies), was dissolved in acidified alcohol at a final concentration of 5 mm and stored at ?20 C. Acidified alcohol was prepared by adding 200 l of 3N HCl to 10 ml of ethanol. Aliquots were dissolved in BRET buffer immediately before use and added to cell suspension at a final concentration of 5 m. BRET measurements were made using a microplate reader (POLARstar Omega, BMG Labtech) equipped with two emission photomultiplier tubes, allowing us CI994 (Tacedinaline) to detect two emissions simultaneously with the highest possible resolution of 50 milliseconds for every data point. All measurements were performed at room temperature. The BRET signal is determined by calculating the ration of the light emitted by G12-Venus (535 nm) over the light emitted by masGRK3ct-Rluc8 (475 nm). The average base-line value (basal R) recorded prior to agonist stimulation was subtracted from BRET signal values, and the resulting difference (R) was normalized against the maximal R value (Rmax) recorded upon agonist stimulation. The rate constants (1/) of the activation and deactivation phases were obtained by fitting a single exponential curve to the traces. for 1 h at 4 C. The detergent-soluble extracts were incubated with nickel-nitrilotriacetic acid beads for 30 CI994 (Tacedinaline) min at 4 C, washed five times with wash buffer (20 mm HEPES (pH 8.0), 380 mm NaCl, 5 mm MgCl2, 0.1% (w/v) C12E10, 20 mm imidazole, 3 mm dithiothreitol, protease inhibitors, 10 m GDP and AlF4? (20 m AlCl3 plus 10 mm NaF) and eluted with SDS sample buffer. CRE-Luciferase Reporter Gene Assays HEK293T/17 cells were transfected with CRE-luc2P reporter (Promega), MOR, Gi1, G1 and G2, or G5 with or without R7 RGS at a 1:1:1:1:1:1:1 ratio between CI994 (Tacedinaline) cDNA constructs using Lipofectamine LTX reagent in 96-well plate. 16 h after transfection, cells were treated with 50.