Prolonged exposure of an agonist to a receptor leads to decreased sensitivity of the receptor to a subsequent agonist challenge. Several distinct mechanisms are involved in this process, for example, uncoupling of receptors from G proteins and reduction in the number of cell surface receptors by internalization or desensitization.81 This phenomenon controls the initiation and termination of the signal and regulates the intensity of response. An early event in the desensitization process is the phosphorylation of the activated receptor by G protein-coupled receptor kinase (GRK) followed by binding of p-arrestin to the phosphorylated receptor. Binding of p-arrestin to the receptor sterically precludes coupling between the receptor and G protein leading to signal termination. p-arrestin acts as an adapter protein that targets GPCRs to clathrin-coated pits for endocytosis. Some GPCRs disappear from cell surfaces within minutes of ligand occupancy. Once internalized, they exhibit distinct patterns of interaction with p-arrestins.
Class A GPCRs, for example, the p2 adrenergic receptors, rapidly dissociate from p-arrestin and are immediately recycled to the plasma membrane. For some GPCRs, receptor occupancy for minutes to hours results in net loss of receptors from cells. For example, angiotensin II receptors form stable receptor-p-arrestin complexes that are accumulated in the endocytic vesicles for degradation or recycling to membranes.82
The Transfluor® technology from Norak Biosciences (Morrisville, North Carolina) is based on labeling p-arrestin with GFP so that the recycling of the recep-tor-arrestin complex can be monitored. Because desensitization occurs only with an activated receptor, monitoring of arrestin translocation within a cell provides a fluorescent bioassay to screen for GPCR ligands. The GFP-p-arrestin is evenly distributed throughout the cytoplasm in the absence of receptor activation. In response to stimulation, the translocation of p-arrestin to the membrane occurs within seconds followed by the movement to endocytic vesicles within minutes.
The application of high content screening to Transfluor technology allows one to screen agonists and antagonists in this format.83-85 The Transfluor technology has been validated on various platforms such as the INCell Analyzer (Amersham Biosciences), Acumen Explorer (TTP Lab Tech, Royston, United Kingdom), ArrayScan (Cellomics, Pittsburgh, Pennsylvania), Discovery-1 (Molecular Devices) and Opera (Evotec OAI, Hamburg, Germany). The application of Transfluor technology has also been validated for a variety of GPCRs.
Bioluminescence resonance energy (BRET) assays have also been reported as translocation techniques for GPCRs.8687 The receptor is fused to renilla luciferase (Rluc) and p-arrestin is fused to GFP. The p-arrestin moiety interacts with the phosphorylated receptor such that Rluc is brought into close proximity with GFP. This results in efficient energy transfer between Rluc and GFP; BRET is measured at 515/410 nm wavelength.
Another generic format for receptor internalization is outlined below. CypHer® is a pH-sensitive dye for receptor internalization studies (CypHer 5 is nonfluorescent at neutral pH and fluorescent at acidic pH). When GPCRs internalize upon agonist binding and activation, the pH changes from neutral at the cell surface to acidic in the intracellular vesicles. Therefore, receptor internalization can be measured as an increase in the fluorescent signal from the cell. The CypHer dye can be coupled to any receptor antibody.88 The antibody bound to the probe is internalized with the receptor and becomes fluorescent in the endocytotic vesicles. Receptor internalization studies are under development and not yet commonly used in HTS.
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