title: Colorimetric assay for rapid screening of corticotropin releasing factor receptor ligands.
abstract: The corticotropin releasing factor (CRF) receptor is known to be coupled to Gs and transduces its signal through stimulation of cyclic AMP (cAMP) production. Here we describe the characterization of several stable CRF receptor-expressing LVIP2.0Zc cell lines that also contain an exogenous cAMP-responsive beta-galactosidase reporter gene construct. The CRF receptor activity was assayed by measuring the induction of beta-galactosidase in response to CRF. Rat/human and bovine CRF stimulated beta-galactosidase activity in a dose-dependent manner with EC50 values of approximately 0.1 nM; the biologically weak deamidated analog of bovine CRF was approximately 500-fold less potent. The CRF receptor antagonist, [d-Phe12,Nle21,38,Ala32]r/hCRF(12-41) produced a dose-dependent inhibition of CRF-stimulated beta-galactosidase activity, further demonstrating the pharmacological specificity of the interaction. The magnitude of the maximal response to CRF varied among individual cell lines. This variation was independent of the level of CRF receptor expression, but reflected differences in the intrinsic activity of adenylate cyclase. In contrast to most cAMP assay systems, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine decreased the CRF-induced beta-galactosidase activity when used in the context of the assay regimen described here. Since the assay can be easily performed in a high-throughput 96-well plate format, these cell lines provide an efficient way for the identification of CRF receptor agonists and antagonists.
authors.affiliation: Neurocrine Biosciences, Inc., San Diego, CA 92121, USA.
authors.forename: C W
authors.initials: CW
authors.lastname: Liaw
authors.forename: D E
authors.initials: DE
authors.lastname: Grigoriadis
authors.forename: E B
authors.initials: EB
authors.lastname: De Souza
authors.forename: T
authors.initials: T
authors.lastname: Oltersdorf
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D008403:Mass Screening / Q000379:methods*; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000502:physiology*; D001616:beta-Galactosidase
chemical_list: D008024:Ligands; D018019:Receptors, Corticotropin-Releasing Hormone; D005576:Colforsin; D000242:Cyclic AMP; D001616:beta-Galactosidase; D015056:1-Methyl-3-isobutylxanthine