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.

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“…We employed a flow cytometric assay of yeast cell binding to and internalization by HEK-293 transfectants. Yeast cells were labeled with the pH-sensitive dye CypHer5E, which increases dramatically in emission intensity after internalization within acidic phagosomal compartments (43,44). The CypHer5E signal was used to measure binding and internalization of yeast cells.…”

Identification of the Low Density Lipoprotein (LDL) Receptor-related Protein-1 Interactome in Central Nervous System Myelin Suggests a Role in the Clearance of Necrotic Cell Debris

“…Necrotic Jurkat cells were labeled with CypHer5, a pH-sensitive fluorescent dye (41), and added to CFSE-labeled LRP1 positive and deficient fibroblasts. After 2 h of incubation at 37°C, engulfment of necrotic cells was analyzed by flow cytometry.…”

A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor γ-chain complex

“…This dye is nonfluorescent at neutral pH, but its fluorescence increases exponentially as it passes through the acidic environment of endosomes and lysosomes, with maximum fluorescence at pH 5, the luminal lysosomal pH (29,30). After CypHer5E-labeled cF1232 was i.v.…”

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