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Updated: Jun 18, 2026

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding
Published on: June 9, 2017
Method for identifying ligands that bind to cloned G(s)- or G(i)-coupled receptors
M König1, L C Mahan, J W Marsg
1Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892, USA.
Abstract:
We describe a fast, simple assay for testing ligands for binding to an unknown receptor after transfection of that receptor cDNA into tissue culture cells. The assay is based on a mouse L cell line (LVIP2.OZc) that contains a cyclic AMP responsive reporter construct and is performed in 96-well plates. If the appropriate agonist binds to a receptor clone coupled to G(s) proteins, activation of adenylyl cyclase produces cAMP which in turn induces the enzyme beta-galactosidase in LVIP2.0Zc cells. beta-Galactosidase activity is detected by staining cells with a chromogenic substrate. After cell lysis, incubation with o-nitrophenyl beta-d-galactopyranoside (ONPG) results in a yellow color. Color development can be observed with the naked eye or read with a plate reader at 405 nm. Agonists that bind to G(i)-coupled receptors can be identified by inhibition of forskolin-induced expression of beta-galactosidase.
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