A sensitive calcium fluorescence assay was developed for the characterization of receptor expression in transformed insect cells. The Drosophila melanogaster genome was utilized in previous research for the isolation of pheromone biosynthesis activating neuropeptide (PBAN)-like G protein-coupled receptors from Helicoverpa zea with stable expression in Spodoptera frugiperda (Sf9) cells. It is known that PBAN receptors will trigger the influx of extracellular calcium. The fluorescent indicator, Fluo-4/AM, was used to detect the influx of calcium after ligand-receptor binding. Fluo-4/AM is cell permeable and is cleaved by an esterase once inside the cell. The de-esterfied Fluo-4/AM may then bind to free calcium and fluoresce. Sf9 cells, loaded with Fluo-4/AM and that were properly expressing the PBAN receptor, were found to fluoresce when PBAN was introduced. A binding curve and the effective concentration were calculated based on the relative fluorescence intensity produced by cells expressing the PBAN receptor.
Species 1: Lepidoptera Noctuidae Helicoverpa zea (corn earworm)
Keywords: fluorescence assay, PBAN receptor
Back to Student Competition Display Presentations, Section B. Physiology, Biochemistry, Toxicology, and Molecular Biology
Back to Student Competition Posters
Back to The 2003 ESA Annual Meeting and Exhibition