Research Advances in Heterotrimeric G-Protein α Subunits and Uncanonical G-Protein Coupled Receptors in Plants

As crucial signal transducers, G-proteins and G-protein-coupled receptors (GPCRs) have attracted increasing attention in the field of signal transduction. Research on G-proteins and GPCRs has mainly focused on animals, while research on plants is relatively rare. The mode of action of G-proteins is quite different from that in animals. The G-protein α (Gα) subunit is the most essential member of the G-protein signal cycle in animals and plants. The G-protein is activated when Gα releases GDP and binds to GTP, and the relationships with the GPCR and the downstream signal are also achieved by Gα coupling. It is important to study the role of Gα in the signaling pathway to explore the regulatory mechanism of G-proteins. The existence of a self-activated Gα in plants makes it unnecessary for the canonical GPCR to activate the G-protein by exchanging GDP with GTP. However, putative GPCRs have been found and proven to play important roles in G-protein signal transduction. The unique mode of action of G-proteins and the function of putative GPCRs in plants suggest that the same definition used in animal research cannot be used to study uncanonical GPCRs in plants. This review focuses on the different functions of the Gα and the mode of action between plants and animals as well as the functions of the uncanonical GPCR. This review employs a new perspective to define uncanonical GPCRs in plants and emphasizes the role of uncanonical GPCRs and Gα subunits in plant stress resistance and agricultural production.     

Function and Regulation of Heterotrimeric G Proteins during Chemotaxis

Chemotaxis, or directional movement towards an extracellular gradient of chemicals, is necessary for processes as diverse as finding nutrients, the immune response, metastasis and wound healing. Activation of G-protein coupled receptors (GPCRs) is at the very base of the chemotactic signaling pathway. Chemotaxis starts with binding of the chemoattractant to GPCRs at the cell-surface, which finally leads to major changes in the cytoskeleton and directional cell movement towards the chemoattractant. Many chemotaxis pathways that are directly regulated by Gβγ have been identified and studied extensively; however, whether Gα is just a handle that regulates the release of Gβγ or whether Gα has its own set of distinct chemotactic effectors, is only beginning to be understood. In this review, we will discuss the different levels of regulation in GPCR signaling and the downstream pathways that are essential for proper chemotaxis.     

Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca²⁺ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we discuss the main mechanisms of GPCR-mediated cell-surface receptors transactivation and the pathways involved in intracellular responses induced by GPCR agonists. These studies may suggest the design of novel strategies for therapeutic interventions.     

Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca²⁺ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gα_q led to a Gα_q-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT₁R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT₁R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gα_q-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.     

Scaffolding of Mitogen-Activated Protein Kinase Signaling by β-Arrestins

β-arrestins were initially identified to desensitize and internalize G-protein-coupled receptors (GPCRs). Receptor-bound β-arrestins also initiate a second wave of signaling by scaffolding mitogen-activated protein kinase (MAPK) signaling components, MAPK kinase kinase, MAPK kinase, and MAPK. In particular, β-arrestins facilitate ERK1/2 or JNK3 activation by scaffolding signal cascade components such as ERK1/2-MEK1-cRaf or JNK3-MKK4/7-ASK1. Understanding the precise molecular and structural mechanisms of β-arrestin-mediated MAPK scaffolding assembly would deepen our understanding of GPCR-mediated MAPK activation and provide clues for the selective regulation of the MAPK signaling cascade for therapeutic purposes. Over the last decade, numerous research groups have attempted to understand the molecular and structural mechanisms of β-arrestin-mediated MAPK scaffolding assembly. Although not providing the complete mechanism, these efforts suggest potential binding interfaces between β-arrestins and MAPK signaling components and the mechanism for MAPK signal amplification by β-arrestin-mediated scaffolding. This review summarizes recent developments of cellular and molecular works on the scaffolding mechanism of β-arrestin for MAPK signaling cascade.     

Fusion with Promiscuous Gα16 Subunit Reveals Signaling Bias at Muscarinic Receptors

A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G₁₆ protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα₁₆ limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα₁₆ mediated pathway more specifically. Our data demonstrated agonist-specific activation of G₁₆ pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα₁₆ pathway at the M₂ receptor and at the same time impaired Gα₁₆ signaling of iperoxo at M₅ receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.     

Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.     

Multiple Mechanisms Mediate Resistance to Sorafenib in Urothelial Cancer

Genetic and epigenetic changes in the mitogen activated protein kinase (MAPK) signaling render urothelial cancer a potential target for tyrosine kinase inhibitor (TKI) treatment. However, clinical trials of several TKIs failed to prove efficacy. In this context, we investigated changes in MAPK signaling activity, downstream apoptotic regulators and changes in cell cycle distribution in different urothelial cancer cell lines (UCCs) upon treatment with the multikinase inhibitor sorafenib. None of the classical sorafenib targets (vascular endothelial growth factor receptor 1/-receptor 2, VEGFR1/-R2; platelet-derived growth factor receptor α/-receptor β, PDGFR-α/-β; c-KIT) was expressed at significant levels leaving RAF proteins as its likely molecular target. Low sorafenib concentrations paradoxically increased cell viability, whereas higher concentrations induced G1 arrest and eventually apoptosis. MAPK signaling remained partly active after sorafenib treatment, especially in T24 cells with an oncogenic HRAS mutation. AKT phosphorylation was increased, suggesting compensatory activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Sorafenib regularly down regulated the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) protein, but combinatorial treatment with ABT-737 targeting other B-cell lymphoma 2 (Bcl-2) family proteins did not result in synergistic effects. In summary, efficacy of sorafenib in urothelial cancer cell lines appears hampered by limited effects on MAPK signaling, crosstalk with further cancer pathways and an anti-apoptotic state of UCCs. These observations may account for the lack of efficacy of sorafenib in clinical trials and should be considered more broadly in the development of signaling pathway inhibitors for drug therapy in urothelial carcinoma.     

Constitutive,Basal, and β-Alanine-Mediated Activation of the Human Mas-Related G Protein-Coupled Receptor D Induces Release of the Inflammatory Cytokine IL-6 and Is Dependent on NF-κB Signaling

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist β-alanine, we observed a release of IL-6. β-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the Gαq/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-κB) and stimulation of IL-6 release. This IL-6 release could be blocked by a Gαq inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for β-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay.     

In Situ PD-L1 Expression in Oral Squamous Cell Carcinoma Is Induced by Heterogeneous Mechanisms among Patients

The expression of programmed death ligand-1 (PD-L1) is controlled by complex mechanisms. The elucidation of the molecular mechanisms of PD-L1 expression is important for the exploration of new insights into PD-1 blockade therapy. Detailed mechanisms of the in situ expression of PD-L1 in tissues of oral squamous cell carcinomas (OSCCs) have not yet been clarified. We examined the mechanisms of PD-L1 expression focusing on the phosphorylation of downstream molecules of epidermal growth factor (EGF) and interferon gamma (IFN-γ) signaling in vitro and in vivo by immunoblotting and multi-fluorescence immunohistochemistry (MF-IHC), respectively. The in vitro experiments demonstrated that PD-L1 expression in OSCC cell lines is upregulated by EGF via the EGF receptor (EGFR)/PI3K/AKT pathway, the EGFR/STAT1 pathway, and the EGFR/MEK/ERK pathway, and by IFN-γ via the JAK2/STAT1 pathway. MF-IHC demonstrated that STAT1 and EGFR phosphorylation was frequently shown in PD-L1-positive cases and STAT1 phosphorylation was correlated with lymphocyte infiltration and EGFR phosphorylation. Moreover, the phosphorylation pattern of the related molecules in PD-L1-positive cells differed among the cases investigated. These findings indicate that PD-L1 expression mechanisms differ depending on the tissue environment and suggest that the examination of the tissue environment and molecular alterations of cancer cells affecting PD-L1 expression make it necessary for each patient to choose the appropriate combination drugs for PD-1 blockade cancer treatment.     

Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for β-Arrestin Recruitment and Receptor Internalization

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of β-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused β-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr²⁷⁹ of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in β-arrestin-mediated MRGPRX2 regulation, we replaced Tyr²⁷⁹ in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced β-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by β-arrestins and that a highly conserved tyrosine residue within MRGPRX2’s NPxxY motif contributes to both G protein- and β-arrestin-mediated responses.     

βArrestins in Cardiac G Protein-Coupled Receptor Signaling and Function: Partners in Crime or “Good Cop,Bad Cop”?

βarrestin (βarr)-1 and -2 (βarrs) (or Arrestin-2 and -3, respectively) are universal G protein-coupled receptor (GPCR) adapter proteins expressed abundantly in extra-retinal tissues, including the myocardium. Both were discovered in the lab of the 2012 Nobel Prize in Chemistry co-laureate Robert Lefkowitz, initially as terminators of signaling from the β-adrenergic receptor (βAR), a process known as functional desensitization. They are now known to switch GPCR signaling from G protein-dependent to G protein-independent, which, in the case of βARs and angiotensin II type 1 receptor (AT₁R), might be beneficial, e.g., anti-apoptotic, for the heart. However, the specific role(s) of each βarr isoform in cardiac GPCR signaling and function (or dysfunction in disease), remain unknown. The current consensus is that, whereas both βarr isoforms can desensitize and internalize cardiac GPCRs, they play quite different (even opposing in certain instances) roles in the G protein-independent signaling pathways they initiate in the cardiovascular system, including in the myocardium. The present review will discuss the current knowledge in the field of βarrs and their roles in GPCR signaling and function in the heart, focusing on the three most important, for cardiac physiology, GPCR types (β₁AR, β₂AR & AT₁R), and will also highlight important questions that currently remain unanswered.     

Structural Characterization of Receptor–Receptor Interactions in the Allosteric Modulation of G Protein-Coupled Receptor (GPCR) Dimers

G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. With the growing influence of computational methods in the study of GPCRs, we will also be reviewing recent computational tools that have been utilized to map protein–protein interactions (PPI).     

G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling

A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)–AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K–AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted.     

Linear Ubiquitination Mediates EGFR-Induced NF-κB Pathway and Tumor Development

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including the NF-κB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-κB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that plakophilin 2 (PKP2) and the linear ubiquitin chain assembly complex (LUBAC) were required for EGFR-mediated NF-κB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IκB phosphorylation and subsequent NF-κB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-κB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-κB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR and that perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy.     

Dominant Role of PI3K p110α over p110β in Insulin and β-Adrenergic Receptor Signalling

Attribution of specific roles to the two ubiquitously expressed PI 3-kinase (PI3K) isoforms p110α and p110β in biological functions they have been implicated, such as in insulin signalling, has been challenging. While p110α has been demonstrated to be the principal isoform activated downstream of the insulin receptor, several studies have provided evidence for a role of p110β. Here we have used isoform-selective inhibitors to estimate the relative contribution of each of these isoforms in insulin signalling in adipocytes, which are a cell type with essential roles in regulation of metabolism at the systemic level. Consistent with previous genetic and pharmacological studies, we found that p110α is the principal isoform activated downstream of the insulin receptor under physiological conditions. p110α interaction with Ras enhanced the strength of p110α activation by insulin. However, this interaction did not account for the selectivity for p110α over p110β in insulin signalling. We also demonstrate that p110α is the principal isoform activated downstream of the β-adrenergic receptor (β-AR), another important signalling pathway in metabolic regulation, through a mechanism involving activation of the cAMP effector molecule EPAC1. This study offers further insights in the role of PI3K isoforms in the regulation of energy metabolism with implications for the therapeutic application of selective inhibitors of these isoforms.     

p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

Saturated stearic acid (SA) induces apoptosis in the human pancreatic β-cells NES2Y. However, the molecular mechanisms involved are unclear. We showed that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway in these cells. Therefore, we tested the role of p38 MAPK signaling pathway activation in apoptosis induction by SA in NES2Y cells. Crosstalk between p38 MAPK pathway activation and accompanying ERK pathway inhibition after SA application was also tested. The inhibition of p38 MAPK expression by siRNA silencing resulted in a decrease in MAPKAPK-2 activation after SA application, but it had no significant effect on cell viability or the level of phosphorylated ERK pathway members. The inhibition of p38 MAPK activity by the specific inhibitor SB202190 resulted in inhibition of MAPKAPK-2 activation and noticeable activation of ERK pathway members after SA treatment but in no significant effect on cell viability. p38 MAPK overexpression by plasmid transfection produced an increase in MAPKAPK-2 activation after SA exposure but no significant influence on cell viability or ERK pathway activation. The activation of p38 MAPK by the specific activator anisomycin resulted in significant activation of MAPKAPK-2. Concerning the effect on cell viability, application of the activator led to apoptosis induction similar to application of SA (PARP cleavage and caspase-7, -8, and -9 activation) and in inhibition of ERK pathway members. We demonstrated that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway and that this activation could be involved in apoptosis induction by SA in the human pancreatic β-cells NES2Y. However, this involvement does not seem to play a key role. Crosstalk between p38 MAPK pathway activation and ERK pathway inhibition in NES2Y cells seems likely. Thus, the ERK pathway inhibition by p38 MAPK activation does not also seem to be essential for SA-induced apoptosis.     

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

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.     

Early Gβγ-GRK2 Inhibition Ameliorates Osteoarthritis Development by Simultaneous Anti-Inflammatory and Chondroprotective Effects

The G-protein-coupled receptor kinase 2 (GRK2) is an important regulator of inflammation and pathological macrophage phenotype in a variety of diseases. We hypothesize that Gβγ-GRK2 signaling promotes the early inflammatory response and chondrocyte loss in osteoarthritis (OA). Using the destabilization of the medial meniscus (DMM) model in 12-week-old male C57BL/6 mice, we determined the role of Gβγ-GRK2 signaling in synovitis, macrophage activation, and OA development. We achieved Gβγ-GRK2 inhibition at the time of DMM by administering the Gβγ inhibitor “gallein” and the GRK2 inhibitor “paroxetine” daily, starting from 2 days before DMM surgery, for a duration of 1 or 12 weeks. Synovial and cartilage structural changes were evaluated by histomorphometry, and molecular events and macrophage activation were examined. We studied the direct role of Gβγ-GRK2 in synovitis and macrophage activation in vitro using SW982 and THP1 cells. Continuous Gβγ-GRK2 inhibition initiated at the time of DMM attenuated OA development and decreased chondrocyte loss more effectively than delayed treatment. GRK2 expression and the M1 macrophage phenotype were elevated in the inflamed synovium, while early gallein and paroxetine treatment for 1 and 12 weeks following DMM resulted in their reduction and an upregulated M2 macrophage phenotype. In vitro experiments showed that Gβγ-GRK2 inhibition attenuated synoviocyte inflammation and the M1 phenotype. We show that early Gβγ-GRK2 inhibition is of higher therapeutic efficacy in OA than delayed inhibition, as it prevents OA development by inhibiting the early inflammatory response.