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Colorimetric assay for rapid screening of corticotropin releasing factor receptor ligands

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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 β-galactosidase reporter gene construct. The CRF receptor activity was assayed by measuring the induction of β-galactosidase in response to CRF. Rat/human and bovine CRF stimulated β-galactosidase activity in a dose-dependent manner with EC50 values of ∼0.1 nM; the biologically weak deamidated analog of bovine CRF was ∼500-fold less potent. The CRF receptor antagonist, [d-Phe12,Nle21,38,Ala32r/hCRF(12–41) produced a dose-dependent inhibition of CRF-stimulated β-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 β-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.

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Authors and Affiliations

  1. Neurocrine Biosciences, Inc., 92121, San Diego, CA

    C. W. Liaw, D. E. Grigoriadis, E. B. De Souza & T. Oltersdorf

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  1. C. W. Liaw
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  2. D. E. Grigoriadis
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  3. E. B. De Souza
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  4. T. Oltersdorf
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Liaw, C.W., Grigoriadis, D.E., De Souza, E.B. et al. Colorimetric assay for rapid screening of corticotropin releasing factor receptor ligands. J Mol Neurosci 5, 83–92 (1994). https://doi.org/10.1007/BF02736750

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  • DOI: https://doi.org/10.1007/BF02736750

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