title: Method for identifying ligands that bind to cloned G(s)- or G(i)-coupled receptors.
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.
authors.affiliation: Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892, USA.
authors.forename: M
authors.initials: M
authors.lastname: König
authors.forename: L C
authors.initials: LC
authors.lastname: Mahan
authors.forename: J W
authors.initials: JW
authors.lastname: Marsg
authors.forename: J S
authors.initials: JS
authors.lastname: Fink
authors.forename: M J
authors.initials: MJ
authors.lastname: Brownstein