sb-408124 and 3-(2-hydroxy-4-(1-1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol

Many G protein-coupled receptors internalize following agonist binding. The studies were designed to identify novel means to effectively quantify this process using the orexin OX(1) receptor and the cannabinoid CB(1) receptor as exemplars.. The human OX(1) and CB(1) receptors were modified to incorporate both epitope tags and variants (SNAP and CLIP) of the enzyme O(6)-alkylguanine-DNA-alkyltransferase within their extracellular, N-terminal domain. Cells able to regulate expression of differing amounts of these constructs upon addition of an antibiotic were developed and analysed.. Cell surface forms of each receptor construct were detected by both antibody recognition of the epitope tags and covalent binding of fluorophores to the O(6)-alkylguanine-DNA-alkyltransferase variants. Receptor internalization in response to agonists but not antagonists could be monitored by each approach but sensitivity was up to six- to 10-fold greater than other approaches when employing a novel, time-resolved fluorescence probe for the SNAP tag. Sensitivity was not enhanced, however, for the CLIP tag, possibly due to higher levels of nonspecific binding.. These studies demonstrate that highly sensitive and quantitative assays that monitor cell surface CB(1) and OX(1) receptors and their internalization by agonists can be developed based on introduction of variants of O(6)-alkylguanine-DNA-alkyltransferase into the N-terminal domain of the receptor. This should be equally suitable for other G protein-coupled receptors.

Topics: Alkyl and Aryl Transferases; Benzoxazoles; Cell Line; Cell Membrane; Cloning, Molecular; Cyclohexanols; Extracellular Signal-Regulated MAP Kinases; Humans; Intracellular Signaling Peptides and Proteins; Ligands; Naphthyridines; Neuropeptides; Orexin Receptors; Orexins; Phenylurea Compounds; Phosphorylation; Piperidines; Plasmids; Pyrazoles; Pyrrolidines; Receptor, Cannabinoid, CB1; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Rimonabant; Thiazoles; Urea