Abstract
The G-protein-coupled receptor (GPCR) regulated intracellular signaling pathway is known to be involved in the development of insecticide resistance in the mosquito, Culex quinquefasciatus. To elucidate the specific role of each effector in the GPCR regulating pathway, we initially expressed a GPCR, G-protein alpha subunit (Gαs), adenylate cyclase (AC), and protein kinase A (PKA) in insect Spodoptera frugiperda (Sf9) cells and investigated their regulation function on cyclic AMP (cAMP) production and PKA activity. GPCR, Gαs, and AC individually expressed Sf9 cells showed higher cAMP production as the expression of each effector increased. All the effector-expressed cell lines showed increased PKA activity however. Moreover, Sf9 cytochrome P450 gene expression and cell tolerance to permethrin were examined. The relative expression of CYP9A32gene in Sf9 cells tested was significantly increased in all effector-expressed cell lines compared to a control cell line; these effector-expressed cell lines also showed significantly higher tolerance to permethrin. Inhibitor treatments on each effector-expressed cell line revealed that Bupivacaine HCl and H89 2HCl robustly inhibited cAMP production and PKA activity, respectively, resulting in decreased tolerance to permethrin in all cell lines. The synergistic functions of Bupivacaine HCl and H89 2HCl with permethrin were further examined in Culex mosquito larvae, providing a valuable new information for mosquito control strategies.
Highlights
G-protein-coupled receptors (GPCRs) are cell surface, membrane-binding proteins that are responsible for signal transmission through extracellular signal binding to activate and regulate intracellular factors
This study focuses on the expression of the mosquito GPCR, Gαs, adenylate cyclase (AC), and protein kinase A (PKA) in insect Sf9 cells via baculovirus-mediated insect expression in order to investigate the specific function of each effector in insecticide resistance and the P450-expressed regulation of insect cells, as well as their complex connection via second messenger and PKA activity
In the Gαs006458 expressed cell line, the cyclic AMP (cAMP) concentrations significantly increased from 12 pmol/mL/mg protein (MOI = 0.2) to 17 pmol/mL/mg protein (MOI = 1) (Figure 1); the same was true for the AC007240 expression cell line, where cAMP concentrations significantly increased from 11 to 14 pmol/mL/mg protein following multiplicity of infection (MOI) increase from 0.2 to 1 (Figure 1)
Summary
G-protein-coupled receptors (GPCRs) are cell surface, membrane-binding proteins that are responsible for signal transmission through extracellular signal binding to activate and regulate intracellular factors. Both the constitutive and spontaneous activities of GPCRs are critically involved in cell signaling responses [1], providing useful opportunities for receptor pharmacology research [2,3]. Many studies have confirmed that GPCRs play a crucial role in regulating insect physiological processes such as development, behavior, metabolism, and reproduction. These conserved intracellular pathways are present in several insect species. Because of the importance of functional GPCRs [8] and their unique fingerprint sequences [9], they have often been considered as potential targets for environmentally friendly insecticides for pest control [10]
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