In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.
advertising feature an8 | December 2008 | nature methods application notes cell Biology biotinylated antibody bound to streptavidin-coated donor beads and a second antibody conjugated to AlphaLISA acceptor beads. The binding of the two antibodies to the analyte brings donor and acceptor beads into proximity. Laser irradiation of donor beads at 680 nm generates a flow of singlet oxygen, triggering a cascade of chemical events in nearby acceptor beads, which results in a chemiluminescent emission at 615 nm. In competitive AlphaLISA immunoassays, a biotinylated analyte bound to streptavidin donor beads is used with an antibody conjugated to AlphaLISA acceptor beads. Rapid and simple quantification of analytesAlphaLISA assays are performed following simple 'mix-and-measure' protocols with reduced hands-on and total assay times compared to ELISAs (Fig. 2). Homogeneous AlphaLISA assays eliminate the need for multiple washes to separate bound from unbound assay components. Miniaturization and automationMiniaturization is a key consideration for reducing screening cost and increasing throughput during the drug-discovery process.AlphaLISA assays are truly miniaturizable and automatable, with ELISA is the most widely used detection platform for the quantification of analytes in biological samples. Because they require multiple washes, ELISAs are difficult to adapt to high throughput and automation. Their relatively narrow dynamic range often requires testing more than one sample dilution. There is clearly a need for simple and more robust alternatives for the quantification of biomarkers in a high-throughput screening format. The new AlphaLISA platform has been specifically designed for that purpose for both the research and drug-discovery fields.The AlphaLISA bead-based technology relies on PerkinElmer's exclusive amplified luminescent proximity homogeneous assay (AlphaScreen ® ) and uses a luminescent oxygen-channeling chemistry 1 . AlphaLISA protocols can be set up as sandwich or competition immunoassays. In a sandwich assay (Fig. 1), an analyte is captured by a AlphaLISA immunoassays: the no-wash alternative to ELISAs for research and drug discovery PerkinElmer's bead-based AlphaLISA® immunoassays are designed for the detection of analytes in biological samples. These chemiluminescent, no-wash assays are ideally suited for miniaturization and automation. They exhibit remarkable sensitivity, wide dynamic range and robust performance that compares advantageously with conventional enzyme-linked immunosorbent assay (ELISA).
Allosteric models of G protein-coupled receptors predict that G protein influences the spontaneous isomerization between inactive (R) and active (R*) conformations. Since inverse agonists have been proposed to preferentially bind to the inactive and uncoupled form(s), changes in the G protein content should influence the binding properties of these ligands. To test this hypothesis, we systematically assessed the effect of G proteins on the binding of beta(2)-adrenergic ligands with distinct levels of inverse efficacy. Recombinant baculoviruses encoding the human beta(2)-adrenoreceptor (beta(2)AR) were expressed alone or in combination with G protein subunits in Sf9 cells. Coexpression with the G protein alpha s beta 1 gamma 2 did not influence the relative efficacy of the ligands to inhibit the adenylyl cyclase but induced considerable decrease in number of sites detected by [(3)H]ICI 118551, [(3)H]propranolol, and (125)I-cyanopindolol. This loss was proportional to the inverse efficacy of the ligand used as the radiotracer in the assay. The addition of Gpp(NH)p inhibited the effects of G protein overexpression indicating that the G proteins acted allosterically. Consistent with this notion, Western blot analysis revealed that coexpression with the G proteins was not accompanied by a loss of immunoreactive beta(2)AR. Such allosteric effects of the G proteins were also observed in mammalian cells expressing endogenous level of G proteins indicating that the phenomenon is not unique to overexpression systems. Taken together, these results demonstrate that the apparent receptor number detected by radiolabeled inverse agonists is affected by the content in G proteins as a result of their influence on R/R* isomerization.
'Corresponding authorThe ability of insulin to promote phosphorylation of the human 02-adrenergic receptor (32AR) was assessed in Chinese hamster fibroblasts transfected with 02AR cDNA. Phosphotyrosine residues were detected in purified j2AR using a polyclonal anti-phosphotyrosine antibody and by phosphoamino acid analysis following metabolic labelling with inorganic 32P. Treatment of the cells with insulin induced a 2.4-fold increase in the phosphotyrosine content of the receptor. The insulinpromoted phosphorylation of the 02AR was accompanied by an increase in the 0-adrenergic-stimulated adenyl cyclase activity. Substitution of a phenylalanine residue for tyrosine-141 completely prevented both the increased tyrosine phosphorylation and the enhanced responsiveness of the ,2AR promoted by insulin treatment. Mutation of three other tyrosines located in the cytoplasmic domain of the receptor, tyrosine-366, tyrosine-350 and tyrosine-354, did not abolish the insulin-promoted tyrosine phosphorylation. Taken together, these results suggest that insulin promotes phosphorylation of the ,B2AR on tyrosine-141 and that such phosphorylation leads to a supersensitization of the receptor.
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