The first reporter gene assay on living cells: green fluorescent protein as reporter gene for the investigation of Gi-protein coupled receptors.
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.
Dinger
Michaela C
MC
Beck-Sickinger
Annette G
AG
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D009478:Neuropeptide Y / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D017476:Receptors, Neuropeptide Y / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D014316:Tritium
D008164:Luminescent Proteins; D009478:Neuropeptide Y; D011956:Receptors, Cell Surface; D017476:Receptors, Neuropeptide Y; C100568:neuropeptide Y5 receptor; D014316:Tritium; D049452:Green Fluorescent Proteins; D005576:Colforsin; D000242:Cyclic AMP