title: CypHer 5: a generic approach for measuring the activation and trafficking of G protein-coupled receptors in live cells.
abstract: GPCRs are one of the most popular classes of therapeutic drug targets. It is therefore important to design specific assay formats to readily identify ligands at these receptors. CypHer 5 technology utilizes the general ability of GPCRs to be internalized into the endosomal pathway of a cell in response to agonist ligands. The CypHer 5 dye is fluorescent in acidic environments, but nonfluorescent at neutral pH. When CypHer 5 is bound to a receptor on the extracellular surface of the cell, it is essentially nonfluorescent. On internalization into a cell, it displays a significant increase in fluorescence. Here we demonstrate the detection of agonist activation of two GPCRs in stably transfected live cells using CypHer 5 technology. The G(q)-coupled TRHR-1 and the G(s)-coupled beta(2)-adrenoceptor were both N-terminally tagged with VSV-G. Following addition of CypHer 5-labeled anti-VSV-G antibodies to HEK 293 cells stably expressing the beta(2)-adrenoceptor or CHO-K1 cells stably expressing the TRHR-1, the cells were treated with agonists and then imaged on Amersham Biosciences' IN Cell Analyzer 3000. Data were quantified using a granularity analysis module. Concentration-response curves were obtained with signal-to-background ratios of 7:1 for both receptors. An EC(50) of 0.52 nM was observed on TRH stimulation of the TRHR-1, and an EC(50) of 30 nM was obtained on isoprenaline stimulation of the beta(2)-adrenoceptor. These results demonstrated that the CypHer technology was capable of measuring high-potency agonist responses. The beta(2)-adrenoceptor antagonist, alprenolol, competed for isoprenaline with an IC(50) of 30 nM, indicating that a high-potency antagonist inhibition curve could also be observed using CypHer. CypHer 5 provides a generic tool to measure GPCR activation in a live cell, homogeneous assay format, and may be equally suitable for detecting activation of other classes of cell surface receptors.
authors.affiliation: Bioassays, Development, Amersham Biosciences, Whitchurch, Cardiff, United Kingdom. Elaine.Adie@uk.amershambiosciences.com
authors.forename: Elaine J
authors.initials: EJ
authors.lastname: Adie
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Francis
authors.forename: June
authors.initials: J
authors.lastname: Davies
authors.forename: Lynne
authors.initials: L
authors.lastname: Smith
authors.forename: Angela
authors.initials: A
authors.lastname: Marenghi
authors.forename: Catherine
authors.initials: C
authors.lastname: Hather
authors.forename: Karen
authors.initials: K
authors.lastname: Hadingham
authors.forename: N Paul
authors.initials: NP
authors.lastname: Michael
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Stephen
authors.initials: S
authors.lastname: Game
mesh_terms: D000818:Animals; D016466:CHO Cells; D002232:Carbocyanines / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D002468:Cell Physiological Phenomena / Q000187:drug effects*; D006224:Cricetinae; D008954:Models, Biological; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects* / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
chemical_list: D002232:Carbocyanines; C474471:CypHer 5 NHS ester; D043562:Receptors, G-Protein-Coupled