G-Protein Coupled Receptors (GPCRs): Signaling Pathways, Characterization, and Functions in Insect Physiology and Toxicology

Abstract

G-protein-coupled receptors (GPCRs) are known to play central roles in the physiology of many organisms. Members of this seven α-helical transmembrane protein family transduce the extracellular signals and regulate intracellular second messengers through coupling to heterotrimeric G-proteins, adenylate cyclase, cAMPs, and protein kinases. As a result of the critical function of GPCRs in cell physiology and biochemistry, they not only play important roles in cell biology and the medicines used to treat a wide range of human diseases but also in insects' physiological functions. Recent studies have revealed the expression and function of GPCRs in insecticide resistance, improving our understanding of the molecular complexes governing the development of insecticide resistance. This article focuses on the review of G-protein coupled receptor (GPCR) signaling pathways in insect physiology, including insects' reproduction, growth and development, stress responses, feeding, behaviors, and other physiological processes. Hormones and polypeptides that are involved in insect GPCR regulatory pathways are reviewed. The review also gives a brief introduction of GPCR pathways in organisms in general. At the end of the review, it provides the recent studies on the function of GPCRs in the development of insecticide resistance, focusing in particular on our current knowledge of the expression and function of GPCRs and their downstream regulation pathways and their roles in insecticide resistance and the regulation of resistance P450 gene expression. The latest insights into the exciting technological advances and new techniques for gene expression and functional characterization of the GPCRs in insects are provided.

Keywords: G-protein coupled receptor regulation pathway; GPCR downstream effectors; cell lines; functional characterization; insect physiology; insecticide resistance.

Figure 1

Graphical representation of the locations of GPCR regulatory pathways. GPCRs can be activated by a variety of ligands, interacting with heterotrimeric G-proteins composed of three subunits (Gα, Gβ, and Gγ) [95], and activating several downstream effector molecules [95]. Gα subunits are classified into four subfamilies: Gαs, Gαi, Gαq, and Gα_12/13 [99,100] and can activate adenylyl cyclase (AC) [101], cyclic adenosine monophosphate (cAMP) [102,103,104], cAMP regulated proteins such as protein kinase A (PKA or cAMP-dependent protein kinase) [105], cyclic nucleotide-gated channels [106], and others [102,104,107], initiating and coordinating intracellular signaling pathways. Gαq can also activate phospholipase C (PLC), which can cleave phosphatidylinositol bisphosphate (PIP₂) into diacylglycerol and inositol triphosphate (IP₃) and membrane-bound diacylglycerol (DAG) [104,108,109,110]. IP3 can open the channel on the endoplasmic reticulum membrane [111], and DAG can activate protein kinase C [104,110]. Within the Gα_12/13 family, G-α₁₃ can increase the activation of p115RhoGEF (the Rho guanine nucleotide exchange factor) and related RhoGEF proteins linked to the Rho activation [112]. Several proteins are known to interact with Gα₁₂, including Btk-family tyrosine kinase, Ras GTPase activating protein, cadherins, p120-caterin, and others [95,104,113,114,115,116,117]. Gβγ subunits can also send signals to phospholipase C, voltage gated Ca²⁺ channels, and others.

Figure 2

The proposed GPCR regulatory pathways in insect physiology processes.

Figure 3

A hypothetical model of the G-protein-coupled receptor (GPCR) intracellular cascade in the insecticide resistance of insects, according to the hypothetical pathway constructed for GPCRs in human cells [95] and in mosquitoes [49,50,51]. The constitutive expressed GPCR in resistant mosquitoes activates the G-protein alpha s-subunit (Gαs), which stimulates adenylate cyclase to convert ATP to cAMP. cAMP activates the protein kinase A, which is involved in the increased expression of cytochrome P450 genes [49,50], resulting in elevating the detoxification ability of insects to insecticides. Inhibitions of cAMP production or PKA activity can interrupt this regulation pathway; the decreased production of cAMP or PKA activity is strongly associated with the decreased expression of resistance-related P450 genes and increased sensitivity to insecticides in both mosquitoes [49,50].

Figure 4

Functional study of GPCR-leading regulation pathway via recombinant baculovirus-GPCR or GFP expression in Sf9 cells. A rhodopsin-like GPCR from the Culex mosquito or a GFP gene was constructed in pENTR^TM plasmid and then recombined with BaculoDirect Linear DNA to form a recombinant baculovirus of GPCR or GFP, expressed in insect Sf9 cells. The recombinant baculovirus gene in the Phase 1 (P1) stage serves as the stock solution for virus amplification in Phase 2 (P2), in which GFP expression visually indicates that cells are alive and active, and thus suitable for use in gene functional studies [174]. The P2 virus continues to be amplified to Phase 3 (P3), which is retained as the final stock solution. A hypothesized GPCR-leading intracellular pathway has been determined for the recombinant virus-GPCR expression cells in P2. In these GPCR expression cells, PKA activity, cAMP production, and Sf9 cell P450 gene expression can be examined after treatment with PKA activity inhibitor (H89 2HCl) or cAMP production inhibitor (Bupivacaine) for comparison with non-treated cells. Declining PKA activity and cAMP concentration, and the decreased expression of P450 genes confirm the involvement of the GPCR-leading pathway in insecticide resistance in vitro.

Figure 5

Graphical representation of the current status of GPCR research on insects and GPCRs and their regulatory pathways in insect physiology.