Abstract
Reporter gene assay systems are important tools for the investigation of G-protein coupled receptors and their interaction with ligands. Here, we describe a novel reporter gene assay system for the investigation of Gi-protein coupled receptors in living cells. For the first time green fluorescent protein (GFP) was used as reporter gene under the transcriptional control of cAMP-response elements (CREs). Three different reporter gene vectors with increasing numbers of CREs were cloned and GFP expression was investigated after forskolin stimulation. Furthermore, the novel reporter system was successfully applied to the neuropeptide Y (NPY) rY5 receptor subtype, a Gi-protein coupled receptor. Our data clearly demonstrate dose-dependent GFP expression for NPY. Furthermore, receptor subtype selectivity of well characterized NPY analog could be proven for the NPY rY5 receptor. The great advantage of the method is that no cell lysis is required and assays can be performed on living cells. Accordingly, in vitro testing of agonist gets faster and significantly more convenient.
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References
Beck-Sickinger, A. G. (1996) Structural characterization and binding sites of G-protein-coupled receptors. Drug Discov. Today 1, 502–513.
Edwards, S. W., Tan, C. M., and L.E., L. (2000) Localisation of G-protein-coupled receptors in health and disease. Trends Pharmacol. Sci. 21, 304–308.
Sautel, M. and Milligan, G. (2000) Molecular manipulation of G-protein-coupled receptors: a new avenue into drug discovery. Curr. Med. Chem. 7, 889–896.
Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S., and Kricka, L. J. (1994) Chemiluminescent and bioluminescent reporter gene assays. Anal. Biochem. 219, 169–181.
Wood, K. V. (1995) Marker proteins for gene expression. Curr. Opin. Biotechnol. 6, 50–58.
Schenborn, E. and Groskreutz, D. (1999) Reporter gene vectors and assays. Mol. Biotechnol. 13, 29–44.
Karin, M. (1994) Signal transduction from the cell surface to the nucleus through the phosphorylation of transcription factors. Curr. Opin. Cell. Biol. 6, 415–424.
Beckers, T., Reilander, H., and Hilgard, P. (1997) Characterization of gonadotropin-releasing hormone analogs based on a sensitive cellular luciferase reporter gene assay. Anal. Biochem. 251, 17–23.
Himmler, A., Stratowa, C., and Czernilofsky, A. P. (1993) Functional testing of human dopamine D1 and D5 receptors expressed in stable cAMP-responsive luciferase reporter cell lines. J. Recept. Res. 13, 79–94.
Stratowa, C., Machat, H., Burger, E., et al. (1995) Functional characterization of the human neurokinin receptors NK1, NK2, and NK3 based on a cellular assay system. J. Recept. Signal Transduct. Res. 15, 617–630.
Migeon, J. C. & Nathanson, N. M. (1994) Differential regulation of cAMP-mediated gene transcription by m1 and m4 muscarinic acetylcholine receptors. Preferential coupling of m4 receptors to Gi alpha-2. J. Biol. Chem. 269, 9767–9773.
Montminy, M. R., Gonzalez, G. A., and Yamamoto, K. K. (1990) Regulation of cAMP-inducible genes by CREB. Trends Neurosci. 13, 184–188.
Ward, W. W. and Cormier, M. J. (1979) An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein. J. Biol. Chem. 254, 781–788.
Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233.
Tsien, R. Y. (1998) The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.
Tannahill, D., Bray, S., and Harris, W. A. (1995) A Drosophila E(spl) gene is “neurogenic” in Xenopus: a green fluorescent protein study. Dev. Biol. 168, 694–697.
Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., and Tsien, R. Y. (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455.
Michel, M. C., Beck-Sickinger, A., Cox, H., et al. (1998) XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol. Rev. 50, 143–150.
Blomqvist, A. G., and Herzog, H. (1997) Y-receptor subtypes-how many more? Trends Neurosci. 20, 294–298.
Cabrele, C. and Beck-Sickinger, A. (2000) Molecular characterisation of the ligand-receptor interaction of the Neuropeptide Y family. J. Peptide Sci. 6, 97–122.
Eckard, C. P., Cabrele, C., Wieland, H. A., and Beck-Sickinger, A. G. (2001) Molecular characterisation of NPY receptors using synthetic peptides and anti-receptor antibodies. Molecules 6, 448–467.
Eckard, C. P., Beck-Sickinger, A. G., and Wieland, H. A. (1999) Comparison of antibodies directed against receptor segments of NPY-receptors. J. Rec. Research and Sign. Transd. 19, 379–394.
Cabrele, C., Langer, M., Bader, R., et al. (2000) The first selective agonist for the neuropeptide YY5 receptor increases food intake in rats. J. Biol. Chem. 275, 36043–36048.
Fuhlendorff, J., Gether, U., Aakerlund, L., et al. (1990) [Leu31, Pro34] Neuropeptide Y: A specific Y1 receptor agonist. Proc. Natl. Acad. Sci. USA 87, 182–186.
Gerald, C., Walker, M. W., Criscione, L., et al. (1996) A receptor subtype involved in neuropeptide-Y-induced food intake. Nature 382, 168–171.
Goetz, A. S., Andrews, J. L., Littleton, T. R., and Ignar, D. M. (2000) Development of a facile method for high throughput screening with reporter gene assays. J. Biomol. Screen. 5, 377–384.
Naylor, L. H. (1999) Reporter gene technology: the future looks bright. Biochem. Pharmacol. 58, 749–757.
Habener, J. F., Miller, C. P., and Vallejo, M. (1995) cAMP-dependent regulation of gene transcription by cAMP response element-binding protein and cAMP response element modulator. Vitam. Horm. 51, 1–57.
de Souza, N. J., Dohadwalla, A. N., and Reden, J. (1983) Forskolin: a labdane diterpenoid with antihypertensive, positive inotropic, platelet aggregation inhibitory, and adenylate cyclase activating properties. Med. Res. Rev. 3, 201–219.
Montmayeur, J. P. and Borrelli, E. (1991) Transcription mediated by a cAMP-responsive promoter element is reduced upon activation of dopamine D2 receptors. Proc. Natl. Acad. Sci. U S A 88, 3135–3139.
Xing, H., Tran, H., Knapp, T. E., Negulescu, P. A., and Pollok, B. A. (2000) A fluorescent reporter assay for the detection of ligands acting through Gi protein-coupled receptors. J. Recept. Signal Transduct. Res. 20, 189–210.
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Dinger, M.C., Beck-Sickinger, A.G. The first reporter gene assay on living cells. Mol Biotechnol 21, 9–18 (2002). https://doi.org/10.1385/MB:21:1:009
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DOI: https://doi.org/10.1385/MB:21:1:009