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Articles by Naiming Zhou in JoVE
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Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy
Jingwen Yang1, Yunjun Yan1, Xiaowei Xiang1, Yuchao Xu1, Naiming Zhou2, Tianming Wang1
1National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College, Zhejiang Ocean University, 2Institute of Biochemistry, College of Life Sciences, Zhejiang University
This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.
Other articles by Naiming Zhou on PubMed
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Cell-cell Fusion and Internalization of the CNS-based, HIV-1 Co-receptor, APJ
Virology.
Mar, 2003 |
Pubmed ID: 12667811 APJ, a member of the human G protein-coupled seven-transmembrane receptor family, has been shown to serve as a coreceptor for the entry of human immunodeficiency virus type I (HIV-1) and simian immunodeficiency virus (SIV), and it is dramatically expressed in central nervous system (CNS)-based cells. In this study, expression of APJ tagged with the green fluorescent protein (GFP) and a fluorescent peptide, 5-carboxyfluorescein (5-CF) conjugated Apelin-13, were utilized for studying receptor internalization and recycling, in stably expressing indicator cells, human neurons, primary CNS microvascular endothelial cells (MVECs), and astrocytes. Fusion of the C-terminus of APJ to the N-terminus of GFP did not alter receptor ligand binding and functions, including signaling and internalization. Using 293 cells stably expressing APJ-GFP, we demonstrated that rapid internalization of the APJ receptor was induced by stimulation with Apelin-36 and Apelin-13, in a dose-dependent manner. Furthermore, investigations showed that the internalized APJ was colocalized with transferrin receptors, suggesting that the internalization of APJ induced by Apelin is likely to be via clathrin-coated pits. Interestingly, we found that the internalized APJ molecules were recycled to the cell surface within 60 min after removal of Apelin-13, but most of the internalized APJ still remained in the cytoplasm, even 2 h after washout of Apelin-36. The intact cytoplasmic C-terminal domain was found to be required for ligand-induced APJ internalization. Human neurons were dramatically stained by the APJ-binding fluorescent peptides. Primary human fetal astrocytes were less strongly labeled with 5-CF-Apelin-13, and in primary human CNS MVECs only weak distribution of green fluorescence specific for APJ in the cytoplasm was observed. Apelin-36 blocked cell membrane fusion mostly due to steric interference, with only a very modest effect on receptor internalization. The CNS represents a unique reservoir site for HIV-1. As such, molecular therapeutics and small molecular inhibitors of HIV-1 entry via this unique CNS receptor are now able to be rationally designed.
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Binding of ALX40-4C to APJ, a CNS-based Receptor, Inhibits Its Utilization As a Co-receptor by HIV-1
Virology.
Jul, 2003 |
Pubmed ID: 12890632 APJ, a G protein-coupled seven-transmembrane receptor, has been shown to serve as a co-receptor for the entry of human immunodeficiency virus type 1 (HIV-1), and it is dramatically expressed in central nervous system (CNS)-based cells. ALX40-4C was identified as a small-molecule antagonist of the chemokine receptor CXCR4, which can specifically inhibit HIV-1 entry via this co-receptor. In this study, we demonstrated that ALX40-4C inhibited both APJ- and CXCR4/APJ-mediated cell membrane fusion in a dose-dependent manner. In competitive binding assays, (125)I-Apelin13 was replaced by ALX40-4C with an IC(50) of 2.9 microM, as compared with an IC(50) of 0.2 nM for Apelin13. Furthermore, ALX40-4C could block ligand-induced APJ internalization and signaling. ALX40-4C, as an antagonist to APJ, directly binds to and prevents use of APJ as a HIV-1 co-receptor. Thus, ALX-4C has potential utility for further elucidation of HIV-1 neuropathogenesis and therapy of HIV-1-induced encephalopathy.
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The N-terminal Domain of APJ, a CNS-based Coreceptor for HIV-1, is Essential for Its Receptor Function and Coreceptor Activity
Virology.
Dec, 2003 |
Pubmed ID: 14675627 The human APJ, a G protein-coupled seven-transmembrane receptor, has been found to be dramatically expressed in the human central nervous system (CNS) and also to serve as a coreceptor for the entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). Studies with animal models suggested that APJ and its natural ligand, apelin, play an important role in the central control of body fluid homeostasis, and in regulation of blood pressure and cardiac contractility. In this study, we characterize the structural and functional determinants of the N-terminal domain of APJ in interactions with its natural ligand and HIV-1 envelope glycoprotein. We demonstrate that the second 10 residues of the N-terminal domain of APJ are critical for association with apelin, while the first 20 amino acids play an important role in supporting cell-cell fusion mediated by HIV-1 gp120. With site-directed mutagenesis, we have identified that the negatively charged amino acid residues Glu20 and Asp23 are involved in receptor and coreceptor functions, but residues Tyr10 and Tyr11 substantially contribute to coreceptor function for both T-tropic (CXCR4) and dual-tropic (CXCR4 and CCR5) HIV-1 isolates. Thus, this study provides potentially important information for further characterizing APJ-apelin functions in vitro and in vivo and designing small molecules for treatment of HIV-1 infection in the CNS.
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A DEAD Box Protein Facilitates HIV-1 Replication As a Cellular Co-factor of Rev
Virology.
Dec, 2004 |
Pubmed ID: 15567440 HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as "bait". DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev- cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics.
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An Enzyme-linked Immunosorbent Assay to Compare the Affinity of Chemical Compounds for β-amyloid Peptide As a Monomer
Analytical and Bioanalytical Chemistry.
Mar, 2010 |
Pubmed ID: 20135308 Aβ(1-42) is the proteolytic cleavage product of cleavage of the amyloid precursor protein by β- and γ-secretases. The aggregation of Aβ(1-42) plays a causative role in the development of Alzheimer's disease. To lock Aβ(1-42) in a homogenous state, we embedded the Aβ(1-42) sequence in an unstructured region of Bcl-x(L). Both the N-terminus and the C-terminus of Aβ(1-42) were constrained in the disordered region, whereas the conjunction did not introduce any folding to Aβ(1-42) but maintained the sequence as a monomer in solution. With Bcl-x(L)-Aβ(42), we developed an enzyme-linked immunosorbent assay to compare the affinity of compounds for monomeric Aβ(1-42). Bcl-x(L)-Aβ(42) was coated on a microplate and this was followed by incubation with different concentrations of compounds. Compounds binding to Leu17-Val24 of Aβ(1-42) inhibited the interaction between Bcl-x(L)-Aβ(42) and antibody 4G8. The method can not only reproduce the activities of the reported Aβ(1-42) inhibitors such as dopamine, tannin, and morin but can also differentiate decoy compounds that do not bind to Aβ(1-42). Remarkably, using this method, we discovered a new inhibitor that binds to monomeric Aβ(1-42) and inhibits Aβ(1-42) fibril formation. As the structure of Bcl-x(L)-Aβ(42) monomer is stable in solution, the assay could be adapted for high-throughput screening with a series of antibodies that bind the different epitopes of Aβ(1-42). In addition, the monomeric form of the Aβ(1-42) sequence in Bcl-x(L)-Aβ(42) would also facilitate the identification of Aβ(1-42) binding partners by coimmunoprecipitation, cocrystallization, surface plasmon resonance technology, or the assay as described here.
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Internalization of the Human Nicotinic Acid Receptor GPR109A is Regulated by G(i), GRK2, and Arrestin3
The Journal of Biological Chemistry.
Jul, 2010 |
Pubmed ID: 20460384 Nicotinic acid (niacin) has been widely used as a favorable lipid-lowering drug for several decades, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin. Mechanistic investigations have shown that as a G(i)-coupled receptor, GPR109A inhibits adenylate cyclase activity upon niacin activation, thereby inhibiting free fatty acid liberation. However, the underlying molecular mechanisms that regulate signaling and internalization of GPR109A remain largely unknown. To further characterize GPR109A internalization, we made a construct to express GPR109A fused with enhanced green fluorescent protein (EGFP) at its carboxyl-terminal end. In stable GPR109A-EGFP-expressing HEK-293 cells, GPR109A-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner upon agonist stimulation. GPR109A internalization was completely blocked by hypertonic sucrose, indicating that GPR109A internalizes via the clathrin-coated pit pathway. Further investigation demonstrated that internalized GPR109A was recycled to the cell surface after the removal of agonist, and recycling of the internalized receptors was not blocked by treatment with acidotropic agents, NH(4)Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca(2+) mobilization; it also significantly attenuated agonist-promoted GPR109A internalization. Moreover, RNA interference experiments showed that knockdown of GRK2 (G protein-coupled receptor kinase 2) and arrestin3 expression significantly impaired receptor internalization. Taken together, these results indicate that the agonist-induced internalization of GPR109A receptors is regulated by GRK2 and arrestin3 in a pertussis toxin-sensitive manner and that internalized receptor recycling is independent of endosomal acidification.
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Structural Optimization and Biological Evaluation of Substituted Bisphenol A Derivatives As Beta-amyloid Peptide Aggregation Inhibitors
Journal of Medicinal Chemistry.
Aug, 2010 |
Pubmed ID: 20684593 The aggregation of Abeta is a crucial step in the etiology of Alzheimer's disease. Our previous work showed that Abeta undergoes alpha-helix/beta-sheet intermediate structures during the conformational transition, and an Abeta aggregation inhibitor (1) was discovered by targeting the intermediates. Here, structure optimization toward compound 1 was performed and 34 novel derivatives were designed and synthesized. Nine compounds showed more effective inhibitory activity than the hit compound 1 in ThT fluorescence assay. Among them, compound 43 demonstrated more excellent inhibitory potency, which not only can suppress the aggregation of Abeta but also can dissolve the preformed fibrils as shown by CD spectroscopy, PICUP and AFM assays. Cellular assay indicated that 43 has no toxicity to neuronal cells, moreover, can effectively inhibit Abeta(1-42)-induced neutrotoxicity and increase the cell viability. Together, on the basis of these positive results, these novel chemical structures may provide a promising potential for therapeutic applications in AD and other types of neurodegenerative disorders.
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The G Protein-coupled Receptors in the Silkworm, Bombyx Mori
Insect Biochemistry and Molecular Biology.
Aug, 2010 |
Pubmed ID: 20685615 G protein-coupled receptors (GPCRs) are the largest and most versatile family of transmembrane receptors in the cell, occupying the highest hierarchical positions in the regulation of many physiological processes. Although they have been extensively studied in a number of model insects, there have been few investigations of GPCRs in large Lepidopterans, such as Bombyx mori, an organism that provides a means to perform detailed tissue expression analyses, which may help to characterize GPCRs and their ligands. In addition, B. mori, also known as the silkworm, is an insect of substantial economic importance, due to its use in silk production and traditional medicines. In this work, we computationally identified 90 putative GPCRs in B. mori, 33 of which represent novel proteins. These GPCRs were annotated and compared with their homologs in Drosophila melanogaster and Anopheles gambiae. Phylogenetics analyses of the GPCRs from these three insects showed that GPCRs may easily duplicate or disappear during insect evolution, especially in the neuropeptide and protein hormone receptor subfamily. Interestingly, we observed a decrease in the quantity and diversity of the stress-tolerance gene, Methuselah, in B. mori, which may be related to its long history of domestication. Moreover, the presence of many Bombyx-specific GPCRs suggests that neither Drosophila nor Anopheles is good representatives for the GPCRs in the Class Insecta.
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Bombyx Adipokinetic Hormone Receptor Activates Extracellular Signal-regulated Kinase 1 and 2 Via G Protein-dependent PKA and PKC but β-arrestin-independent Pathways
Biochemistry.
Dec, 2010 |
Pubmed ID: 21126059 Neuropeptides of the adipokinetic hormone (AKH) family are among the best studied hormone peptides. They play important roles in insect hemolymph sugar homeostasis, larval lipolysis, and storage-fat mobilization. Mechanistic investigations have shown that, upon AKH stimulation, adipokinetic hormone receptor (AKHR) couples to a Gs protein and enhances adenylate cyclase activity, leading to intracellular cAMP accumulation. However, the underlying molecular mechanism by which this signaling pathway connects to extracellular signal-regulated kinase 1/2 (ERK1/2) remains to be elucidated. Using HEK293 cells stably or transiently expressing AKHR, we demonstrated that activation of AKHR elicited transient phosphorylation of ERK1/2. Our investigation indicated that AKHR-mediated activation of ERK1/2 was significantly inhibited by H-89 (protein kinase A inhibitor), Go6983, and GF109203X (protein kinase C inhibitors) but not by U73122 (PLC inhibitor) or FIPI (PLD inhibitor). Moreover, AKHR-induced ERK1/2 phosphorylation was blocked by the calcium chelators EGTA and BAPTA-AM. Furthermore, ERK1/2 activation in both transiently and stably AKHR-expressing HEK293 cells was found to be sensitive to pretreatment of pertussis toxin, whereas AKHR-mediated ERK1/2 activation was insensitive to siRNA-induced knockdown of β-arrestins and to pretreatment of inhibitors of EGFR, Src, and PI3K. On the basis of our data, we propose that activated AKHR signals to ERK1/2 primarily via PKA- and calcium-involved PKC-dependent pathways. Our current study provides the first in-depth study defining the mechanisms of AKH-mediated ERK activation through the Bombyx AKHR.
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Identification and Characterization of INCB9471, an Allosteric Noncompetitive Small-molecule Antagonist of C-C Chemokine Receptor 5 with Potent Inhibitory Activity Against Monocyte Migration and HIV-1 Infection
The Journal of Pharmacology and Experimental Therapeutics.
Jul, 2011 |
Pubmed ID: 21459966 C-C chemokine receptor 5 (CCR5) is a clinically proven target for inhibition of HIV-1 infection and a potential target for various inflammatory diseases. In this article, we describe 5-[(4-{(3S)-4-[(1R,2R)-2-ethoxy-5-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-3-methylpiperazin-1-yl}-4-methylpiperidin-1-yl)carbonyl]-4,6-dimethylpyrimidine dihydrochloride (INCB9471), a potent and specific inhibitor of human CCR5 that has been proven to be safe and efficacious in viral load reduction in phase I and II human clinical trails. INCB9471 was identified using a primary human monocyte-based radioligand competition binding assay. It potently inhibited macrophage inflammatory protein-1β-induced monocyte migration and infection of peripheral blood mononuclear cells by a panel of R5-HIV-1 strains. The results from binding and signaling studies using incremental amounts of INCB9471 demonstrated INCB9471 as a noncompetitive CCR5 inhibitor. The CCR5 residues that are essential for interaction with INCB9471 were identified by site-specific mutagenesis studies. INCB9471 rapidly associates with but slowly dissociates from CCR5. When INCB9471 was compared with three CCR5 antagonists that had been tested in clinical trials, the potency of INCB9471 in blocking CCR5 ligand binding was similar to those of 4,6-dimethyl-5-{[4-methyl-4-((3S)-3-methyl-4-{(1R0-2-(methyloxy)-1-[4-(trifluoromethyl) phenyl]ethyl}-1-piperazingyl)-1-piperidinyl]carbonyl}pyrimidine (SCH-D; vicriviroc), 4-{[4-({(3R)-1-butyl-3-[(R)-cyclohexyl(hydroxyl)methyl]-2, 5-dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenyl]oxy}benzoic acid hydrochloride (873140; aplaviroc), and 4,4-difluoro-N-((1S)-3-{(3-endo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl}-1-phenylpropyl)cyclohexanecarboxamide (UK427857; maraviroc). Its inhibitory activity against CCR5-mediated Ca(2+) mobilization was also similar to those of SCH-D and 873140. Further analysis suggested that INCB9471 and UK427857 may have different binding sites on CCR5. The significance of two CCR5 antagonists with different binding sites is discussed in the context of potentially overcoming drug-resistant HIV-1 strains.
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Identification of Distinct C-terminal Domains of the Bombyx Adipokinetic Hormone Receptor That Are Essential for Receptor Export, Phosphorylation and Internalization
Cellular Signalling.
Sep, 2011 |
Pubmed ID: 21536126 Neuropeptides of the adipokinetic hormone (AKH) family play important roles in insect hemolymph sugar homeostasis, larval lipolysis and storage-fat mobilization. Our previous studies have shown that the adipokinetic hormone receptor (AKHR), a Gs-coupled receptor, induces intracellular cAMP accumulation, calcium mobilization and ERK1/2 phosphorylation upon agonist stimulation. However, the underlying molecular mechanisms that regulate the internalization and desensitization of AKHR remain largely unknown. In the current study we made a construct to express AKHR fused with enhanced green fluorescent protein (EGFP) at its C-terminal end to further characterize AKHR internalization. In stable AKHR-EGFP-expressing HEK-293 cells, AKHR-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner via the clathrin-coated pit pathway upon agonist stimulation, and internalized receptors were slowly recovered to the cell surface after the removal of AKH peptides. The results derived from RNA interference and arrestin translocation demonstrated that G protein-coupled receptor kinase 2 and 5 (GRK2/5) and β-arrestin2 were involved in receptor phosphorylation and internalization. Furthermore, experiments using deletion and site-directed mutagenesis strategies identified the three residues (Thr356, Ser359 and Thr362) responsible for GRK-mediated phosphorylation and internalization and the C-terminal domain from residue-322 to residue-342 responsible for receptor export from ER. This is the first detailed investigation of the internalization and trafficking of insect G protein-coupled receptors.
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Distinct Kinetic and Spatial Patterns of Protein Kinase C (PKC)- and Epidermal Growth Factor Receptor (EGFR)-dependent Activation of Extracellular Signal-regulated Kinases 1 and 2 by Human Nicotinic Acid Receptor GPR109A
The Journal of Biological Chemistry.
Sep, 2011 |
Pubmed ID: 21768093 Nicotinic acid (niacin) has been widely used as a lipid-lowering drug for several decades, and recently, orphan G protein-coupled receptor GPR109A has been identified as a receptor for niacin. Mechanistic investigations have shown that, upon niacin activation, GPR109A couples to a G(i) protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for GPR109A signaling remain largely unknown. Using CHO-K1 cells stably expressing GPR109A and A431 cells, which are a human epidermoid cell line with high levels of endogenous expression of functional GPR109A receptors, we found that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by niacin was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that GPR109A induced ERK1/2 activation via the matrix metalloproteinase/epidermal growth factor receptor transactivation pathway at both early and later time points (2-5 min); this pathway was distinct from the PKC pathway-mediated ERK1/2 phosphorylation that occurs at early time points (≤2 min) in response to niacin. Overexpression of Gβγ subunit scavengers βARK1-CT and the Gα subunit of transducin led to a significant reduction of ERK1/2 phosphorylation, suggesting a critical role for βγ subunits in GPR109A-activated ERK1/2 phosphorylation. Using arrestin-2/3-specific siRNA and an internalization-deficient GPR109A mutant, we found that arrestin-2 and arrestin-3 were not involved in GPR109A-mediated ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to niacin GPR109A receptors initially activate G(i), leading to dissociation of the Gβγ subunit from activated G(i), and subsequently induce ERK1/2 activation via two distinct pathways, one PKC-dependent pathway occurring at a peak time of ≤2 min and the other matrix metalloproteinase-dependent growth factor receptor transactivation occurring at both early and later time points (2-5 min).
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Identification and Functional Characterization of Two Orphan G-protein-coupled Receptors for Adipokinetic Hormones from Silkworm Bombyx Mori
The Journal of Biological Chemistry.
Dec, 2011 |
Pubmed ID: 22009754 Adipokinetic hormones (AKHs) are the best studied insect neuropeptides with the function of mobilizing lipids and carbohydrates during energy-expensive activities and modulating fundamental physiological processes, such as sugar homeostasis, lipid metabolism, and reproduction. Three distinct cDNAs encoding the prepro-Bombyx AKH1-3 have been cloned and confirmed by mass spectrometric methods. Our previous research suggested the Bombyx AKH receptor is activated by AKH1 and AKH2 with high affinity but by AKH3 with quite low affinity. In this study, using stable functional expression of the receptors in HEK293 cells, we have now identified AKH3 as a specific ligand for two orphan G-protein-coupled receptors, and we therefore named them AKHR2a and AKHR2b, respectively. We demonstrated that both AKHR2a and AKHR2b were activated by AKH3 at high affinity and by AKH1 and AKH2 at low affinity, leading to an increase of intracellular cAMP levels and activation of ERK1/2 and receptor internalization, but they were not activated by Bombyx corazonin. Conversely, the Bombyx corazonin receptor was activated by corazonin but not by AKH1-3. Quantitative RT-PCR revealed that AKHR2a and AKHR2b were both highly expressed in the testis but were also detected at low levels in other tissues. These results will lead to a better understanding of the AKH/AKHR system in the regulation of fundamental physiological processes.
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Searching for the Multi-Target-Directed Ligands Against Alzheimer's Disease: Discovery of Quinoxaline-based Hybrid Compounds with AChE, H₃R and BACE 1 Inhibitory Activities
Bioorganic & Medicinal Chemistry.
Dec, 2011 |
Pubmed ID: 22019465 A novel series of quinoxaline derivatives, as Multi-Target-Directed Ligands (MTDLs) for AD treatment, were designed by lending the core structural elements required for H(3)R antagonists and hybridizing BACE 1 inhibitor 1 with AChE inhibitor BYYT-25. A virtual database consisting of quinoxaline derivatives was first screened on a pharmacophore model of BACE 1 inhibitors, and then filtered by a molecular docking model of AChE. Seventeen quinoxaline derivatives with high score values were picked out, synthesized and evaluated for their biological activities. Compound 11a, the most effective MTDL, showed the potent activity to H(3)R/AChE/BACE 1 (H(3)R antagonism, IC(50)=280.0 ± 98.0 nM; H(3)R inverse agonism, IC(50)=189.3 ± 95.7 nM; AChE, IC(50)=483 ± 5 nM; BACE 1, 46.64±2.55% inhibitory rate at 20 μM) and high selectivity over H(1)R/H(2)R/H(4)R. Furthermore, the protein binding patterns between 11a and AChE/BACE 1 showed that it makes several essential interactions with the enzymes.
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Identification and Characterization of Distinct C-terminal Domains of the Human Hydroxycarboxylic Acid Receptor-2 That Are Essential for Receptor Export, Constitutive Activity, Desensitization, and Internalization
Molecular Pharmacology.
Dec, 2012 |
Pubmed ID: 22962331 The human hydroxycarboxylic acid receptor 2 (HCA₂), also known as GPR109A and HM74a, was first identified as a niacin receptor and has recently received significant attention because of its potential to clinically modify plasma lipids in a favorable manner. Our recent studies have demonstrated that the niacin-induced internalization of HCA₂ receptors is regulated by G protein-coupled receptor kinase (GRK) 2 and arrestin3 and that internalized receptors rapidly recycle back to the cell surface. The investigation presented here used a combination of amino acid deletion and site-directed mutagenesis to identify structural and functional domains within the HCA₂ C terminus and explore their potential roles in receptor phosphorylation, desensitization, and internalization. We first constructed four mutants with deletions of 10 to 15 amino acids each that were distinct from truncated mutants. We successfully identified different domains responsible for receptor export, constitutive activity, desensitization, phosphorylation, and internalization. We also generated a comprehensive series of alanine substitution mutants, replacing conserved serine and threonine residues in the C terminus with alanine residues to pinpoint the key residues that are essential for GRK2-mediated phosphorylation and arrestin3 association. Moreover, we found that a sequence from residues 329 to 343 in the C-terminal tail of HCA₂ plays a crucial role in keeping HCA₂ in an inactive conformation. These data demonstrate the importance of distinct domains within the C terminus of HCA₂ for receptor cell surface expression, desensitization, and internalization and phosphorylation and stabilization of an inactive receptor conformation.
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Specific Activation of the G Protein-coupled Receptor BNGR-A21 by the Neuropeptide Corazonin from the Silkworm, Bombyx Mori, Dually Couples to the G(q) and G(s) Signaling Cascades
The Journal of Biological Chemistry.
Apr, 2013 |
Pubmed ID: 23457297 Corazonin, an undecapeptide neurohormone sharing a highly conserved amino acid sequence across Insecta, plays different physiological roles in the regulation of heart contraction rates, silk spinning rates, the induction of dark color and morphometric phase changes, and ecdysis. Corazonin receptors have been identified in Drosophila melanogaster, Manduca sexta, and Musca domestica. However, detailed information on the signaling and major physiological functions of corazonin and its receptor is largely unknown. In the current study, using both the mammalian cell line HEK293 and insect cell lines BmN and Sf21, we paired the Bombyx corazonin neuropeptide as a specific endogenous ligand for the Bombyx neuropeptide G protein-coupled receptor A21 (BNGR-A21), and we therefore designated this receptor as BmCrzR. Further characterization indicated that synthetic BmCrz demonstrated a high affinity for and activated BmCrzR, resulting in intracellular cAMP accumulation, Ca(2+) mobilization, and ERK1/2 phosphorylation via the Gq- and Gs-coupled signaling pathways. The direct interaction of BmCrzR with BmCrz was confirmed by a rhodamine-labeled BmCrz peptide. Moreover, experiments with double-stranded RNA and synthetic peptide injection suggested a possible role of BmCrz/BmCrzR in the regulation of larval growth and spinning rate. Our present results provide the first in-depth information on BmCrzR-mediated signaling for further elucidation of the BmCrz/BmCrzR system in the regulation of fundamental physiological processes.
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Activation of CAMP-response Element-binding Protein is Positively Regulated by PKA and Calcium-sensitive Calcineurin and Negatively by PKC in Insect
Insect Biochemistry and Molecular Biology.
Nov, 2013 |
Pubmed ID: 24018109 The cAMP response element binding protein, CREB, is a G protein-coupled receptor (GPCR) signal-activated transcription factor implicated in the control of many biological processes. In the current study, we constructed a cAMP response element (CRE)-driven luciferase assay system for GPCR characterization in insect cells. Our results indicated that Gs-coupled Bombyx adipokinetic hormone receptor (AKHR) and corazonin receptor could effectively initiate CRE-driven luciferase transcription, but forskolin, a reagent widely used to activate adenylyl cyclase in mammalian systems, failed to induce luciferase activity in insect cells co-transfected with a CRE-driven reporter construct upon agonist treatment. Further investigation revealed that the specific protein kinase C (PKC) inhibitors exhibited stimulatory effects on CRE-driven reporter transcription, and blockage of Ca(2+) signals and inhibition of Ca(2+)-dependent calcineurin resulted in a significant decrease in the luciferase activity. Taken together, these results suggest that PKC likely acts as a negative regulator to modulate CREB activation; in contrast, Ca(2+) signals and Ca(2+)-dependent calcineurin, in addition to PKA, essentially contribute to the positive regulation of CREB activity. This study presents evidence to elucidate the underlying molecular mechanism by which CREB activation is regulated in insects.
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Activation of Bombyx Neuropeptide G Protein-coupled Receptor A4 Via a Gαi-dependent Signaling Pathway by Direct Interaction with Neuropeptide F from Silkworm, Bombyx Mori
Insect Biochemistry and Molecular Biology.
Feb, 2014 |
Pubmed ID: 24374022 Members of the mammalian neuropeptide Y (NPY) family serve as neurotransmitters and contribute to a diversity of physiological functions. Although neuropeptide F (NPF), the NPY-like orthologs from insects, have been identified, the NPF receptors and their signaling and physiological functions remain largely unknown. In this study, we established the stable and transient functional expression of a Bombyx orphan G protein-coupled receptor, BNGR-A4, in both mammalian HEK293 and insect SF21 cells. We identified Bombyx mori NPFs as specific endogenous ligands for the Bombyx Neuropeptide GPCR A4 (BNGR-A4) and, accordingly, named the receptor BomNPFR. Our results demonstrated that BomNPFR was activated by synthetic BomNPF1a and BomNPF1b at a high efficacy and by BomNPF2 at a low efficacy. This activation led to a decrease of forskolin or adipokinetic hormone peptide-stimulated adenylyl cyclase activity, an increase of intracellular Ca(2+), the activation of ERK1/2 signaling and receptor internalization. Moreover, a Rhodamine-labeled BomNPF1a peptide was found to bind specifically to BomNPFR. The results derived from quantitative RT-PCR analysis and dsRNA-mediated knockdown experiments demonstrated the possible role of BomNPFR in the regulation of food intake and growth. Our results provide the first in-depth information on BomNPFR-mediated signaling for the further elucidation of the BomNPF/BomNPFR system in the regulation of fundamental physiological processes.
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Melatonin Receptor Type 1 Signals to Extracellular Signal-regulated Kinase 1 and 2 Via Gi and Gs Dually Coupled Pathways in HEK-293 Cells
Biochemistry.
May, 2014 |
Pubmed ID: 24724723 The pineal gland hormone melatonin exerts its regulatory roles in a variety of physiological and pathological responses through two G protein-coupled receptors, melatonin receptor type 1 (MT1) and melatonin receptor type 2 (MT2), which have been recognized as promising targets in the treatment of a number of human diseases and disorders. The MT1 receptor was identified nearly 20 years ago; however, the molecular mechanisms by which MT1-mediated signaling affects physiology remain to be further elucidated. In this study, using HEK293 cells stably expressing the human MT1 receptor, melatonin induced a concentration-dependent activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). The melatonin-mediated phosphorylation of ERK1/2 at later time points (≥5 min) was strongly suppressed by pretreatment with pertussis toxin, but only a slight, if any, inhibition of ERK1/2 activation at early time points (≤2 min) was detected. Further experiments demonstrated that the Gβγ subunit, phosphoinositide 3-kinase, and calcium-insensitive protein kinase C were involved in the MT1-mediated activation of ERK1/2 at later time points (≥5 min). Moreover, results derived from cAMP assays combined with a MT1 mutant indicated that the human MT1 receptor could also couple to Gs protein, stimulating intracellular cAMP formation, and that the MT1-induced activation of ERK1/2 at early time points (≤2 min) was mediated by the Gs/cAMP/PKA cascade. Our findings may provide new insights into the pharmacological effects and physiological functions modulated by the MT1-mediated activation of ERK1/2.
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Involvement of β-arrestin-2 and Clathrin in Agonist-mediated Internalization of the Human Cannabinoid CB2 Receptor
Current Molecular Pharmacology.
2014 |
Pubmed ID: 25023974 The CB2 cannabinoid receptor is a promising therapeutic target for the treatment of inflammatory diseases, neuropathic pain, liver diseases, cancer and cardiovascular diseases. Obtaining detailed information on the internalization and trafficking of the human CB2 receptor in response to agonist will have a significant impact on drug discovery. Visualization and quantitative detection of EGFP-tagged CB2 receptor showed that, upon WIN55,212-2 stimulation, the CB2 receptor was rapidly internalized in a dose- and time-dependent manner from the cell membrane into the cytoplasm. Pretreatment with hypertonic sucrose, MDC clathrin inhibitor, or siRNA-mediated knock-down of clathrin heavy chain led to significant inhibition of agonist-induced CB2 internalization. Using the RNA interference method, we showed that knockdown of β-arrestin2 expression significantly impaired receptor internalisation. Further investigation demonstrated that the internalized CB2 receptors were co-localized with the early endosome probe and were recycled to the cell surface after the removal of agonist, but treatment with specific cell-permeable proteasome inhibitor MG132 a inhibited the recycling of internalized CB2 receptor, suggesting that the proteasome-mediated degradation pathway may be involved in CB2 internalization. Moreover, the single residue Ser(352) and residue cluster S(335)S(336)T(338)T(340) at the C-terminal tail are shown to be essential for receptor phosphorylation and β-arrestin2 association. These data provide new insights into the mechanisms regulating agonist-mediated internalization and trafficking of the human CB2 receptor.
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Differential Requirements of Arrestin-3 and Clathrin for Ligand-dependent and -independent Internalization of Human G Protein-coupled Receptor 40
Cellular Signalling.
Nov, 2014 |
Pubmed ID: 25038452 G protein-coupled receptor 40 (GPR40) is believed to be an attractive target to enhance insulin secretion in patients with type 2 diabetes. GPR40 has been found to couple to Gq protein, leading to the activation of phospholipase C and subsequent increases in the intracellular Ca(2+) level. However, the underlying mechanisms that regulate the internalization and desensitization of GPR40 remain to be elucidated. In the present study, a construct of GPR40 fused with enhanced green fluorescent protein (EGFP) at its C-terminus was constructed for direct imaging of the localization and internalization of GPR40 by confocal microscopy. In stably transfected HEK-293 cells, GPR40 receptors underwent rapid agonist-induced internalization and constitutive ligand-independent internalization. Our data demonstrated that the agonist-mediated internalization of GPR40 was significantly blocked by hypertonic sucrose treatment and by siRNA mediated depletion of the heavy chain of clathrin. In contrast, constitutive GPR40 internalization was not affected by hypertonic sucrose or by knock-down of clathrin expression, but it was affected by treatment with methyl-β-cyclodextrin (MβCD) and nystatin. Furthermore, our results using an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3 and GRK2 expression revealed that arrestin-3 and GRK2 play an essential role in the regulation of agonist-mediated GPR40 internalization, but are not involved in the regulation of constitutive GPR40 internalization. Additionally, our observation showed that upon activation by agonist, the internalized GPR40 receptors were rapidly recycled back to the plasma membrane via Rab4/Rab5 positive endosomes, whereas the constitutively internalized GPR40 receptors were recycled back to the cell surface through Rab5 positive endosomes. Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this family.
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Bombyx Mori Prothoracicostatic Peptide Receptor is Allosterically Activated Via a Gα(i/o)-protein-biased Signalling Cascade by Drosophila Sex Peptide
The Biochemical Journal.
Mar, 2015 |
Pubmed ID: 25422933 In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169-31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca²⁺ mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM-DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM-BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gα(i/o)-dependent cAMP production and away from Ca²⁺ mobilization and arrestin recruitment.
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Novel 1-Phenyl-3-hydroxy-4-pyridinone Derivatives As Multifunctional Agents for the Therapy of Alzheimer's Disease
ACS Chemical Neuroscience.
Jan, 2016 |
Pubmed ID: 26479744 A series of novel 1-phenyl-3-hydroxy-4-pyridinone derivatives were designed and synthesized as multifunctional agents for Alzheimer's disease (AD) therapy through incorporation of 3-hydroxy-4-pyridinone moiety from deferiprone into the scaffold of H3 receptor antagonists. Most of these new compounds displayed designed quadruple functions, H3 receptor antagonism, Aβ aggregation inhibition, metal ion chelation, and radical scavenging. Especially, the most promising compound 5c displayed nanomolar IC50 values in H3 receptor antagonism with high selectivity, efficient capability to interrupt the formation of Aβ(1-42) fibrils, good copper and iron chelating properties, and more potent 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) radical cation (ABTS(•+)) scavenging activity than Trolox. Further biological evaluation revealed that it did not show obvious cytotoxicity and hERG potassium channel inhibition at micromolar concentration. In addition, compound 5c demonstrated suitable pharmacokinetic properties and acceptable blood-brain barrier (BBB) permeability in vivo. All these results indicate that compound 5c is a potential multifunctional candidate for AD therapy.
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Agonist-induced Activation of Histamine H3 Receptor Signals to Extracellular Signal-regulated Kinases 1 and 2 Through PKC-, PLD-, and EGFR-dependent Mechanisms
Journal of Neurochemistry.
Apr, 2016 |
Pubmed ID: 26826667 The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here, we provide evidence that upon activation by an agonist, H3Rs trigger ERK1/2 activation via phospholipase C/protein kinase C (PLC/PKC)-, phospholipase D (PLD)s-, and matrix metallopeptidase/epidermal growth factor receptor (MMP/EGFR) transactivation-dependent pathways. Moreover, we demonstrate that H3Rs exhibit a neuroprotective effect on the cultured mouse cortical neurons under hypoxia conditions through the ERK1/2 pathway.
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Human Neuropeptide S Receptor Is Activated Via a Gαq Protein-biased Signaling Cascade by a Human Neuropeptide S Analog Lacking the C-terminal 10 Residues
The Journal of Biological Chemistry.
Apr, 2016 |
Pubmed ID: 26865629 Human neuropeptide S (NPS) and its cognate receptor regulate important biological functions in the brain and have emerged as a future therapeutic target for treatment of a variety of neurological and psychiatric diseases. The human NPS (hNPS) receptor has been shown to dually couple to Gαs- and Gαq-dependent signaling pathways. The human NPS analog hNPS-(1-10), lacking 10 residues from the C terminus, has been shown to stimulate Ca(2+)mobilization in a manner comparable with full-length hNPSin vitrobut seems to fail to induce biological activityin vivo Here, results derived from a number of cell-based functional assays, including intracellular cAMP-response element (CRE)-driven luciferase activity, Ca(2+)mobilization, and ERK1/2 phosphorylation, show that hNPS-(1-10) preferentially activates Gαq-dependent Ca(2+)mobilization while exhibiting less activity in triggering Gαs-dependent CRE-driven luciferase activity. We further demonstrate that both Gαq- and Gαs-coupled signaling pathways contribute to full-length hNPS-mediated activation of ERK1/2, whereas hNPS-(1-10)-promoted ERK1/2 activation is completely inhibited by the Gαqinhibitor UBO-QIC but not by the PKA inhibitor H89. Moreover, the results of Ala-scanning mutagenesis of hNPS-(1-13) indicated that residues Lys(11)and Lys(12)are structurally crucial for the hNPS receptor to couple to Gαs-dependent signaling. In conclusion, our findings demonstrate that hNPS-(1-10) is a biased agonist favoring Gαq-dependent signaling. It may represent a valuable chemical probe for further investigation of the therapeutic potential of human NPS receptor-directed signalingin vivo.
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Quantitative Analysis of G-protein-coupled Receptor Internalization Using DnaE Intein-based Assay
Methods in Cell Biology.
2016 |
Pubmed ID: 26928549 G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, are involved in many physiological processes. They represent highly important therapeutic targets for drug discovery. Currently, there are numerous cell-based assays developed for the pharmacological profiling of GPCRs and the identification of novel agonists and antagonists. However, the development of new, faster, easier, and more cost-effective approaches to detect GPCR activity remains highly desirable. β-arrestin-dependent internalization has been demonstrated to be a common mechanism for most GPCRs. Here we describe a novel assay for quantitative analysis of GPCR internalization based on DnaE intein-mediated reconstitution of fragmented Renilla luciferase or Firefly luciferase when activated GPCRs interact with β-arrestin2 or Rab5. Further validation, using functionally divergent GPCRs, showed that EC50 values obtained for the known agonists and antagonists were in close agreement with the results of previous reports. This suggests that this assay is sensitive enough to permit quantification of GPCR internalization. Compared with conventional assays, this novel assay system is cost-effective, rapid, and easy to manipulate. These advantages may allow this assay to be used universally as a functional cell-based system for GPCR characterization and in the screening process of drug discovery.
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Agonist-mediated Activation of Bombyx Mori Diapause Hormone Receptor Signals to Extracellular Signal-regulated Kinases 1 and 2 Through Gq-PLC-PKC-dependent Cascade
Insect Biochemistry and Molecular Biology.
Aug, 2016 |
Pubmed ID: 27318251 Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter. This unique process is regulated by diapause hormone (DH), which is a neuropeptide hormone that induces egg diapause in Bombyx mori and is involved in terminating pupal diapause in heliothis moths. An G protein-coupled receptor from the silkworm, B. mori, has been identified as a specific cell surface receptor for DH. However, the detailed information on the DH-DHR system and its mechanism(s) involved in the induction of embryonic diapause remains unknown. Here, we combined functional assays with various specific inhibitors to elucidate the DHR-mediated signaling pathways. Upon activation by DH, B. mori DHR is coupled to the Gq protein, leading to a significant increase of intracellular Ca(2+) and cAMP response element-driven luciferase activity in an UBO-QIC, a specific Gq inhibitor, sensitive manner. B. mori DHR elicited ERK1/2 phosphorylation in a dose- and time-dependent manner in response to DH. This effect was almost completely inhibited by co-incubation with UBO-QIC and was also significantly suppressed by PLC inhibitor U73122, PKC inhibitors Gö6983 and the Ca(2+) chelator EGTA. Moreover, DHR-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ specific inhibitors gallein and M119K and the PI3K specific inhibitor Wortmannin, but not by the Src specific inhibitor PP2. Our data also demonstrates that the EGFR-transactivation pathway is not involved in the DHR-mediated ERK1/2 phosphorylation. Future efforts are needed to clarify the role of the ERK1/2 signaling pathway in the DH-mediated induction of B. mori embryonic diapause.
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