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United States Department of Agriculture

Agricultural Research Service

Research Project: ECOLOGICALLY BASED MANAGEMENT OF BOLL WEEVILS AND OTHER ROW CROP PESTS UNDER TRANSITION TO BOLL WEEVIL ERADICATION IN TEMPERATE REGIONS

Location: Insect Control and Cotton Disease Research Unit

Title: Development of cell-based bioassay with SF9 cells expressing TcSKR1 and TcSKR2 and differential activation by sulfated and non-sulfated SK peptides involved in food intake regulation

Authors
item Yu, Na -
item Swevers, Luc -
item Nachman, Ronald
item Smagghe, Guy -

Submitted to: Peptides
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 17, 2014
Publication Date: March 15, 2014
Citation: Yu, N., Swevers, L., Nachman, R.J., Smagghe, G. 2014. Development of cell-based bioassay with SF9 cells expressing TcSKR1 and TcSKR2 and differential activation by sulfated and non-sulfated SK peptides involved in food intake regulation. Peptides. 53:238-242.

Interpretive Summary: Insect pests have developed resistance to several conventional pesticides, and new approaches are needed for pest management. Although neuropeptides (short chains of amino acids) serve as potent messengers in insects to regulate vital functions, the neuropeptides hold little promise as pest control agents because they can be degraded in the target pest. New, selective control agents may be developed by designing mimics of these neuropeptides that resist degradation and either inhibit or over-stimulate critical neuropeptide-regulated life functions. We report on the isolation, identification and characterization of two different active sites for the sulfakinin hormone in the red flour beetle, an important pest of stored grain. One of these two different receptors was found to modulate food intake and appetite, whereas the other had no effect. In beetles and other insects, this hormone regulates the critical functions of food intake and appetite. The work identified a specific chemical structure necessary to turn on the active site. This discovery, and the new information gained from it, will aid in the design of neuropeptide-like compounds capable of disrupting the food intake and/or appetite of these and related moths. This may eventually lead to development of practical neuropeptide-like substances that can effectively control pest insects of in an environmentally friendly fashion.

Technical 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. As major results, western blotting confirmed a stable presence of the two TcSKRs in the transformed cell lines. This system demonstrated that TcSKRs were coupled to G-alpha-s-protein upon activation and stimulated cAMP accumulation. Exposure of the transfected cell lines to sulfated SK (sSK) activated both TcSKRs at 1 nM; the EC50 of sSK to obtain 50% of receptor activation was similar for the two receptors. In contrast, uM concentrations of nonsulfated 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, facilitating an understanding of the mechanism of SK regulation of food intake in insects and providing new insights on the development of new methods to control pest insects.

Last Modified: 9/10/2014
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