The second messenger 3′, 5′-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.
References
1.
Nogrady, T. (1988). Medicinal Chemistry—A Biological approach, 2nd ed, pp. 58-59. Oxford University Press, New York.
2.
Lodish, H., Baltimore, D., Berk, A., Zipursky, S., Matsudaira, P., Darnell, J. (1995). Molecular Cell Biology, 3rd ed, pp. 857-881. Scientific American Books, Inc., New York.
3.
Xiao, Z., Yao, Y., Long, Y., Devreotes, P. (1999). Desensitization of G Protein-coupled receptors. J. Biol. Chem.274:1440-1448.
4.
Barak, L.S., Ferguson, S.S.G., Zhang, J., Caron, M.C. (1997). A β-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation. J. Biol. Chem.272:27497-27500.
5.
Grouzmann, E., Buclin, T., Martire, M., Cannizzaro, C., Dorner, B., Razaname, A., Mutter, M. (1997). Characterization of a selective antagonist of neuropeptide Y at the Y2 receptor. J. Biol. Chem.272:7699-7706.
6.
Ball, H.J., Shine, J., Herzog, H. (1995). Multiple promoters regulate tissue specific expression of the human NPY-Y 1 receptor gene. J. Biol. Chem.270:27272-27276.
7.
Hu, Y., Bloomquist, B.T., Cornfield, L.J., DeCarr, L.B., et al., (1996). Identification of a novel hypothalmic neuropeptide Y receptor associated with feeding behavior. J. Biol. Chem.271:26315-26319.
8.
Wildey, M., Homon, C., Hutchins, B. (1999). Allegro™: Moving the bar upwards. J. Biomol. Screen.4:57-60.
9.
Kariv, I., Stevens, M.E., Behrens, D.L., Oldenburg, K.R. (1999). High throughput quantitation of cAMP production mediated by activation of seven transmembrane domain receptors. J. Biomol. Screen.4(1), pp. 27-32.
10.
FlashPlate File #9. (1998). Color quench correction on FlashPlate. NEN Life Science Products, Boston.
11.
Zhang, J., Chung, T.D.Y., Oldenburg, K.R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen.4:77-73.
12.
Amersham Life Science. (1996). High throughput screening for cAMP formation by scintillation proximity radioimmunoassay. Proximity News, Issue no. 23.
13.
NEN Life Science Products. (1999). Adenylyl cyclase activation FlashPlate assay—A direct cAMP [125I] detection assay (Catalog Number SMPOO4, SMP004A). NEN Life Science Products, Boston.
14.
Kebabian, J.W. (1992). The cyclic AMP cascade: A signal transduction system, Research Biochemicals Inc. Neurotransmissions.8(2):1-4.
15.
Kirby, D.A., Koerber, S.C., May, J.M., Hagaman, C., Cullen, M.J., Pelleymounter, M.A., Rivier, J.E. (1995). Y1 and Y2 receptor selective neuropeptide Y analogues: Evidence for a Y1 receptor subclass. J. Med. Chem.38:4579-4586.
