A green-fluorescent-protein-based assay for the characterization of G-protein-coupled receptors.
A simple and sensitive system to monitor activation and pharmacology of G-protein-coupled receptors in mammalian cells is described. It is based on cAMP-responsible-element-regulated expression of green fluorescent protein (GFP). Cotransfection with appropriate G-protein-coupled receptors and subsequent activation with agonists induces expression of GFP in a dose-dependent manner. This system is suited for the analysis of most G-protein-coupled receptors, including those that are coupled to Gs, Gi, and Gq. It can replace reporter systems for G-protein-coupled receptors currently in use. Time-consuming and labor-intensive analysis is avoided because it is a noninvasive system, which allows multiple reprobing without disturbing the cells. In addition, adaptation to high-throughput approaches is possible. Together with human embryonic kidney cells, it is a zero-background expression system, making it ideally suited to the pharmacological characterization of cloned receptors, to expression cloning experiments, and to the identification of the natural ligand of orphan G-protein-coupled receptors.
Roeder
Thomas
T
Biomedizinisches Forschungszentrum, Phillips Universität Marburg, D-35035 Marburg, Germany. roeder@zoologie.uni-hamburg.de
Görich
Derk
D
Heyden
Dörte
D
Gewecke
Michael
M
D000818:Animals; D019556:COS Cells; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D043562:Receptors, G-Protein-Coupled / Q000032:analysis*; D011970:Receptors, Histamine H2 / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection / Q000379:methods
D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011970:Receptors, Histamine H2