title: Development of cell-based bioassay with Sf9 cells expressing TcSKR1 and TcSKR2 and differential activation by sulfated and non-sulfated SK peptides.
abstract: Insect sulfakinin receptors (SKRs) are G-protein-coupled receptors (GPCRs) that interact with sulfakinins (SKs) to modulate diverse biological processes. One of the indispensable roles of SKs is in the regulation of food intake in insects. In this project we report on the development of a cell-based receptor assay system with insect Sf9 cells, expressing TcSKR1 and TcSKR2 from the red flour beetle Tribolium castaneum, a model and important pest insect in agriculture. In this system, a stable presence of the two TcSKRs was supported by Western blotting. The expressed TcSKRs were coupled to Gαs-protein upon activation and stimulated cAMP accumulation in Sf9 cells. Exposure of the transfected cell lines to sulfated SK (sSK) activated TcSKR1 at 1 nM; the EC50 of sSK to obtain 50% of receptor activation was similar for both receptors. In contrast, μM concentrations of non-sulfated SK were necessary to activate both TcSKRs. In conclusion, this cell-based TcSKR assay system is useful to screen SK-related peptides and mimetics and to better document ligand-receptor structure-activity relationships. Given the importance of SK signaling system in insects, the present study may provide new insights on the development of new methods to control pest insects.
authors.affiliation: Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, 9000 Gent, Belgium.
authors.forename: Na
authors.initials: N
authors.lastname: Yu
authors.affiliation: Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications National Centre for Scientific Research "Demokritos", Athens, Greece.
authors.forename: Luc
authors.initials: L
authors.lastname: Swevers
authors.affiliation: Insect Control and Cotton Disease Research, Southern Plains Agricultural Research Center, USDA, College Station, TX 77845, USA.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Nachman
authors.affiliation: Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, 9000 Gent, Belgium. Electronic address: guy.smagghe@ugent.be.
authors.forename: Guy
authors.initials: G
authors.lastname: Smagghe
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001835:Body Weight / Q000502:physiology; D016466:CHO Cells; D002460:Cell Line; D003412:Cricetulus; D004435:Eating / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D061987:Sf9 Cells
chemical_list: D010455:Peptides
keywords: Cell-based assay; G-protein-coupled receptor; Gαs-protein; Sulfakinin; Sulfakinin receptor