An Essential Role of c-Fos in Notch1-mediated Promotion of Proliferation of KSHV-Infected SH-SY5Y Cells.

BACKGROUND: This study aimed to investigate the influence of Notch1 on c-Fos and the effect of c-Fos on the proliferation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected neuronal cells. METHODS: Real-time PCR and western blotting were used to determine c-Fos expression levels in KSHV-infected (SK-RG) and uninfected SH-SY5Y cells. C-Fos levels were measured again in SK-RG cells with or without Notch1 knockdown. Next, we measured c-Fos and p-c-Fos concentrations after treatment with the Notch1 γ-secretase inhibitor LY-411575 and the Notch1 activator Jagged-1. MTT and Ki-67 staining were used to evaluate the proliferation ability of cells after c-Fos levels downregulation. CyclinD1, CDK6, and CDK4 expression levels and cell cycle were analyzed by western blotting and flow cytometry, respectively. After the c-Fos intervention, the KSHV copy number and gene expression of RTA, LANA and K8.1 were analyzed by real-time TaqMan PCR. RESULTS: C-Fos was up-regulated in KSHV-infected SK-RG cells. However, the siRNA-mediated knockdown of Notch1 resulted in a significant decrease in the levels of c-Fos and p-c-Fos (P <0.01, P <0.001). Additionally, a decrease in Cyclin D1, CDK6, and CDK4 was also detected. The Notch1 inhibitor LY-411575 showed the potential to down-regulate the levels of c-Fos and p-c-Fos, which was consistent with Notch1 knockdown group (P <0.01), whereas the expression and phosphorylation of c-Fos were remarkably up-regulated by treatment of Notch1 activator Jagged-1 (P <0.05). In addition, our data obtained by MTT and Ki-67 staining revealed that the c-Fos down-regulation led to a significant reduction in cell viability and proliferation of the SK-RG cells (P <0.001). Moreover, FACS analysis showed that the cell cycle was arrested in the G0/G1 stage, and the expressions of Cyclin D1, CDK6, and CDK4 were down-regulated in the c-Fos-knockdown SK-RG cells (P <0.05). Reduction in total KSHV copy number and expressions of viral genes (RTA, LANA and K8.1) were also detected in c-Fos down-regulated SK-RG cells (P <0.05). CONCLUSION: Our findings strongly indicate that c-Fos plays a crucial role in the promotion of cell proliferation through Notch1 signaling in KSHV-infected cells. Furthermore, our results suggest that the inhibition of expression of key viral pathogenic proteins is likely involved in this process.

Verifying AXL and putative proteins as SARS-CoV-2 receptors by DnaE intein-based rapid cell-cell fusion assay.

As the understanding of the mechanisms of SARS-CoV-2 infection continues to grow, researchers have come to realize that ACE2 and TMPRSS2 receptors are not the only way for the virus to invade the host, and that there are many molecules that may serve as potential receptors or cofactors. The functionality of these numerous receptors, proposed by different research groups, demands a fast, simple, and accurate validation method. To address this issue, we here established a DnaE intein-based cell-cell fusion system, a key result of our study, which enables rapid simulation of SARS-CoV-2 host cell infection. This system allowed us to validate that proteins such as AXL function as SARS-CoV-2 spike protein receptors and synergize with ACE2 for cell invasion, and that proteins like NRP1 act as cofactors, facilitating ACE2-mediated syncytium formation. Our results also suggest that mutations in the NTD of the SARS-CoV-2 Delta variant spike protein show a preferential selection for Spike-AXL interaction over Spike-LDLRAD3. In summary, our system serves as a crucial tool for the rapid and comprehensive verification of potential receptors, screening of SARS-CoV-2-neutralizing antibodies, or targeted drugs, bearing substantial implications for translational clinical applications.

Melatonin modulates the hypothalamic-pituitary neuroendocrine axis to regulate physiological color change in teleost fish.

Melatonin (MT) is a crucial neuroendocrine regulator of various physiological activities in vertebrates, especially in circadian or seasonal rhythm control. In the present study, the large yellow croaker (Larimichthys crocea), a marine bony fish with circadian body color change behavior, is chosen for functional investigation on teleost MT signaling systems that remain uncharacterized. All five melatonin receptors (LcMtnr1a1, LcMtnr1a2, LcMtnr1b1, LcMtnr1b2, and LcMtnr1c) were significantly activated by MT, triggering extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation through different G protein coupling signaling pathways, with exclusive Gαi-dependency for LcMtnr1a2 and LcMtnr1c, and Gαq-dependency for two LcMtnr1b paralogs, whereas LcMtnr1a1 activated Gαi and Gαs dual-dependent signaling pathways. A comprehensive model of the MT signaling system in the hypothalamic-pituitary neuroendocrine axis was further constructed based on ligand-receptor interaction analysis using single-cell RNA-seq data, as well as spatial expression patterns of Mtnrs and related neuropeptides in central neuroendocrine tissues. A novel regulatory pathway of MT/melanin-concentrating hormone (MCH) and MT/(tachykinin precursor 1 (TAC1)+corticotropin-releasing hormone (CRH))/melanocyte-stimulating hormone (MSH) was discovered that functions in chromatophore mobilization and physiological color change and was further validated by pharmacological experiments. Together, our findings define multiple intracellular signaling pathways mediated by L. crocea melatonin receptors and provide the first in-depth evidence that uncover the upstream modulating roles of the MT signaling system in the hypothalamic-pituitary neuroendocrine axis of a marine teleost species, particularly in chromatophore mobilization and physiological color change.

Two naturally occurring mutations of human GPR103 define distinct G protein selection bias.

The neuropeptide 26RFa plays important roles in the regulation of many physiological functions. 26RFa has been recognized as an endogenous ligand for receptor GPR103. In the present study, we demonstrate that GPR103 dually couples to Gαq and Gαi/o proteins. However, two naturally occurring missense mutations were identified from a young male patient. In the first, Y68H, induction of Ca2+ mobilization was noted without detection of ERK1/2 activation. In the second, R371W, the potential to activate ERK1/2 signaling was retained but with failure to evoke Ca2+ mobilization. Further analysis provides evidence that Gαq, L-type Ca2+ channel and PKCßI and ßII are involved in the Y68H-mediated signaling pathway, whereas Gαi/o, Gßγ, and PKCζ are implicated in the R371W-induced signaling. Our results demonstrate that two point mutations, Y68H and R371W, affect the equilibrium between the different receptor conformations, leading to alteration of G protein-coupling preferences. Importantly, these findings provide a foundation for future elucidation of GPCR-mediated biased signaling and the physiological implications of their bias.

Identification and characterization of a novel 5-hydroxytryptamine receptor in the sea cucumber Apostichopus japonicus (Selenka).

Serotonin (5-hydroxytryptamine [5-HT]) receptors (5-HTRs) mediate neuroendocrine signaling via interactions with the ligand serotonin (5-HT). The 5-HT signaling system has been well studied in vertebrates, but rarely known in invertebrate animals, especially in the marine invertebrates. In this study, we identified and characterized a novel 5-HTR from the sea cucumber Apostichopus japonicus (Aj5-HT4/6 ). The cloned Aj5-HT4/6 open reading frame comprised 1290 bp and encoded 429 amino acids. Bioinformatic analysis of the receptor indicated that it was a member of the class A of the G protein-coupled receptor family. Further experiments using Aj5-HT4/6 -transfected HEK293 cells demonstrated that treatment with 5-HT could induce rapid internalization of Aj5-HT4/6 fused with enhanced green fluorescent protein from the cell surface into the cytoplasm and triggered a significant increase in levels of the second messenger cAMP as well as mitogen-activated protein kinase phosphorylation in a 5-HT dose-dependent manner. Quantitative real time-polymerase chain reaction demonstrated that Aj5-HT4/6 was predominantly expressed in the muscle and respiratory tree, and its expression was significantly decreased during estivation. Taken together, these results imply that Aj5-HT4/6 is potentially involved in the movement and metabolism of the sea cucumber.

N-glycosylation of the human neuropeptide QRFP receptor (QRFPR) is essential for ligand binding and receptor activation.

The newly identified pyroglutamylated RFamide peptide (QRFP) signaling system has been shown to be implicated in regulating a variety of physiological processes. G-protein-coupled receptors (GPCRs) are preferentially N-glycosylated on extracellular domains. The human QRFP receptor QRFPR (GPR103) possesses three N-glycosylation consensus sites, two located on the N-terminal domain (N5 and N19) and one on the first extracellular loop (ECL1) (N106); however, to date, their role in QRFPR expression and signaling has not been established. Here, we combined mutants with glutamine substitution of the critical asparagines of the consensus sites with glycosidase PNGase F and N-glycosylation inhibitor tunicamycin to study the effect of N-glycosylation in the regulation of QRFPR cell surface expression and signaling. Western blot analysis performed with site-directed mutagenesis revealed that two asparagines at N19 in the N-terminus and N106 in ECL1, but not N5 in the N-terminus, served as sites for N-glycosylation. Treatment with PNGase F and tunicamycin resulted in a reduction in both two-protein species, ~43 kDa and ~85 kDa in size, by 2-4 kDa. Analysis with confocal microscopy and quantitative ELISA showed that N-glycosylation of QRFPR is not essentially required for targeting the cell membrane. However, further binding assay and functional assays demonstrated that removal of N-glycosylation sequons or treatment with tunicamycin led to significant impairments in the interaction of receptor with QRFP26 and downstream signaling. Thus, our findings suggest that for the human QRFP receptor (QRFPR), N-glycosylation is not important for cell surface expression but is a pre-requisite for ligand binding and receptor activation.

Bombyx neuropeptide G protein-coupled receptor A14 and A15 are two functional G protein-coupled receptors for CCHamide neuropeptides.

CCHamides are newly identified insect neuropeptides, which are widely occurring in most insects. However, our knowledge about their signaling characteristics and physiological roles is still limited. Here, we cloned two full-length cDNAs encoding putative CCHamide receptors, Bombyx neuropeptide GPCR A14 (BNGR-A14) and -A15 (BNGR-A15), from the brain of B. mori larvae. Characterization of signaling indicated that Bombyx CCHamide-1 and CCHamide-2 are specific endogenous ligands for BNGR-A15 and BNGR-A14, respectively. Further functional assays combined with specific inhibitors demonstrated that upon activation by CCHamide-2, BNGR-A14 elicited significant increases in CRE-driven luciferase activity, intracellular Ca2+ mobilization and ERK1/2 phosphorylation in a Gq inhibitor-sensitive manner, while BNGR-A15 was activated by CCHamide-1, thus leading to intracellular accumulation of cAMP, Ca2+ mobilization, and ERK1/2 phosphorylation in a Gs and Gq inhibitor-sensitive manner. Based on these findings, we designated the receptors BNGR-A15 and -A14 as Bommo-CCHaR-1 and -2, respectively. In addition, our results showed that CCHamides are considered to require intrachain disulfide bonds to activate their respective receptor in the physiological concentration range. Moreover, quantitative RT-PCR analysis revealed that CCHamide-1 is more likely to serve as a brain-gut peptide to regulate feeding behavior and growth through BNGR-A15, whereas the CCHamide-2 signaling system might play an important role in the control of multiple physiological processes. Our findings provide in-depth information on CCHamide-1 and -2-mediated signaling, facilitating further elucidation of their endocrinological roles in the regulation of fundamental physiological processes.

Activation of Bombyx mori neuropeptide G protein-coupled receptor A19 by neuropeptide RYamides couples to Gq protein-dependent signaling pathways.

RYamides constitute a novel family of neuropeptides newly identified in insects, and play important roles in regulating a variety of physiological processes. However, the signaling characteristics and physiological actions of RYamide signaling system remain largely unknown. In the present study, we cloned the full-length complementary DNA of the RYamide receptor BNGR-A19 from Bombyx mori larvae. After expression in mammalian HEK293T and insect Sf9 cells, functional assays revealed that BNGR-A19 was activated by synthetic RYamide peptides, triggering a significant increase in cAMP-response element controlled luciferase activity and Ca2+ mobilization in a Gq inhibitor-sensitive manner. Upon activation by RYamide peptides, BNGR-A19 elicited ERK1/2 phosphorylation via a Gq -PLC-PKC pathway, and also underwent a rapid internalization from the cell surface to the cytoplasm. Further cross-activity analysis indicated that BNGR-A19 exhibited very weak response upon stimulation by high concentration (1 µM) of Bombyx sulfakinin-1, neuropeptide F-1, and short neuropeptide F-1, and vice versa, Bombyx RYamides also showed slight potency for activating Bombyx NPF receptor (BNGR-A4) and sNPF receptor (BNGR-A11). In addition, the quantitative reverse-transcription polymerase chain reaction results showed that the high-level expression of BNGR-A19 was detected in the hindgut and testis, suggesting that the RYamide signaling is likely involved in the regulation of feeding, water homeostasis and testis development. This study provides the first in-depth information on the insect RYamide signaling system, facilitating the further clarification of its endocrinological roles in insect physiology.

Synthesis and antiseizure effect evaluation of nonimidazole histamine H3 receptor antagonists containing the oxazole moiety.

The use of histamine H3 receptor (H3 R) antagonists is becoming a promising therapeutic approach for epilepsy. In this paper, a series of novel nonimidazole H3 R antagonists was synthesized and screened as antiepileptic drugs. All of these prepared antagonists displayed micromolar or submicromolar H3 R antagonistic activities in the cAMP response element luciferase screening assay. Compounds 5a (IC50 = 0.11 µM), 5b (IC50 = 0.56 µM), and 5f (IC50 = 0.78 µM) displayed the most potent H3 R antagonistic activities, with considerable potency when compared with pitolisant (IC50 = 0.51 µM). In the maximal electroshock (MES)-induced seizure model, compounds 5c, 5e, and 5g showed obvious protection for the electrostimulated mice, and the protection of 5g against the MES-induced seizures was fully abrogated when mice were cotreated with R-(α)-methyl-histamine, a central nervous system-penetrant H3 R agonist, suggesting that the potential therapeutic effect of 5g was observed to work through H3 R. These results indicate that the attempt to find a new antiepileptic drug among H3 R antagonists is practicable, but it is necessary to consider the log P of the molecules to ensure penetration of the blood-brain barrier.

Design, synthesis, and anticonvulsant effects evaluation of nonimidazole histamine H3 receptor antagonists/inverse agonists containing triazole moiety.

Histamine H3 receptors (H3R) antagonists/inverse agonists are becoming a promising therapeutic approach for epilepsy. In this article, novel nonimidazole H3R antagonists/inverse agonists have been designed and synthesised via hybriding the H3R pharmacophore (aliphatic amine with propyloxy chain) with the 1,2,4-triazole moiety as anticonvulsant drugs. The majority of antagonists/inverse agonists prepared here exerted moderate to robust activities in cAMP-response element (CRE) luciferase screening assay. 1-(3-(4-(3-Phenyl-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3l) and 1-(3-(4-(3-(4-chlorophenyl)-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3m) displayed the highest H3R antagonistic activities, with IC50 values of 7.81 and 5.92 nM, respectively. Meanwhile, the compounds with higher H3R antagonistic activities exhibited protection for mice in maximal electroshock seizure (MES)-induced convulsant model. Moreover, the protection of 3m against the MES induced seizures was fully abrogated when mice were co-treated with RAMH, a CNS-penetrant H3R agonist, which suggested that the potential therapeutic effect of 3m was through H3R. These results indicate that the attempt to find new anticonvulsant among H3R antagonists/inverse agonists is practicable.

Existence and functions of a kisspeptin neuropeptide signaling system in a non-chordate deuterostome species.

The kisspeptin system is a central modulator of the hypothalamic-pituitary-gonadal axis in vertebrates. Its existence outside the vertebrate lineage remains largely unknown. Here, we report the identification and characterization of the kisspeptin system in the sea cucumber Apostichopus japonicus. The gene encoding the kisspeptin precursor generates two mature neuropeptides, AjKiss1a and AjKiss1b. The receptors for these neuropeptides, AjKissR1 and AjKissR2, are strongly activated by synthetic A. japonicus and vertebrate kisspeptins, triggering a rapid intracellular mobilization of Ca2+, followed by receptor internalization. AjKissR1 and AjKissR2 share similar intracellular signaling pathways via Gαq/PLC/PKC/MAPK cascade, when activated by C-terminal decapeptide. The A. japonicus kisspeptin system functions in multiple tissues that are closely related to seasonal reproduction and metabolism. Overall, our findings uncover for the first time the existence and function of the kisspeptin system in a non-chordate species and provide new evidence to support the ancient origin of intracellular signaling and physiological functions that are mediated by this molecular system.

Functional characterization of neuropeptide 26RFa receptors GPR103A and GPR103B in zebrafish, Danio rerio.

The hypothalamic neuropeptide 26RFa is the most recently identified member of the RFamide peptide family, and this 26RFa signaling system has been shown to be implicated in regulating a variety of physiological processes. In zebrafish，26RFa and two putative receptors, DrGPR103A and DrGPR103B, have been in silico identified, and in vivo data derived from overexpression and loss of function mutation experiments suggest the 26RFa signaling system plays an important role in the hypothalamic regulation of sleep. However, the biochemical and pharmacological information on DrGPR103A/B receptors is still unknown. Here, after cloning of cDNAs of two putative 26RFa receptor genes, DrGPR103A and B, from the total RNA of zebrafish whole body, functional assays demonstrated that both receptors were activated by synthetic zebrafish 26RFa neuropeptide, leading to a significant increase in CRE-driven luciferase activity and intracellular Ca2+ mobilization in a Gαq inhibitor- and Gαi/o inhibitor-sensitive manner. Upon activation by 26RFa, DrGPR103A and B evoked ERK1/2 phosphorylation and underwent internalization. Further functional determination also revealed that zebrafish kisspeptin-1 exhibited a slight potency for activating both DrGPR103A and B, and vice versa, zebrafish 26RFa also showed some activity at zebrafish GPR54A and B. Our findings provided evidence that zebrafish GPR103A and B are two functional Gαq- and Gαi/o-dually coupled receptors for 26RFa, enabling the further elucidation of the endocrinological roles of zebrafish 26RFa signaling system in the regulation of physiological activities.

A novel splice variant of Gαq-coupled Bombyx CAPA-PVK receptor 1 functions as a specific Gαi/o-linked receptor for CAPA-PK.

Alternative splicing enables G protein-coupled receptor (GPCR) genes to greatly increase the number of structurally and functionally distinct receptor isoforms. However, the functional role and relevance of the individual GPCR splice variants in regulating physiological processes are still to be assessed. A naturally occurring alternative splice variant of Bombyx CAPA-PVK receptor, BomCAPA-PVK-R1-Δ341, has been shown to act as a dominant-negative protein to regulate cell surface expression and function of the canonical CAPA-PVK receptor. Herein, using functional assays, we identify the splice variant Δ341 as a specific receptor for neuropeptide CAPA-PK, and upon activation, Δ341 signals to ERK1/2 pathway. Further characterization demonstrates that Δ341 couples to Gαi/o, distinct from the Gαq-coupled canonical CAPA-PVK receptor, triggering ERK1/2 phosphorylation through Gßγ-PI3K-PKCζ signaling cascade. Moreover, our ELISA data show that the ligand-dependent internalization of the splice variant Δ341 is significantly impaired due to lack of GRKs-mediated phosphorylation sites. Our findings highlight the potential of this knowledge for molecular, pharmacological and physiological studies on GPCR splice variants in the future.

Ligand-dependent internalization of Bombyx mori tachykinin-related peptide receptor is regulated by PKC, GRK5 and ß-arrestin2/BmKurtz.

Tachykinin signaling system is present in both vertebrates and invertebrates, and functions as neuromodulator responsible for the regulation of various physiological processes. In human, the internalization of G protein-coupled receptors has been extensively characterized; however, the insect GPCR internalization has been rarely investigated. Here, we constructed two expression vectors of Bombyx tachykinin-related peptide receptor (BmTKRPR) fused with Enhanced Green Fluorescent Protein (EGFP) at the C-terminal end for direct visualization of receptor expression, localization, and trafficking in cultured mammalian HEK293 and insect Sf21 cells. Our results demonstrated that agonist-activated BmTKRPR underwent rapid internalization in a dose-and time-dependent manner via a clathrin-dependent pathway in both HEK293 and Sf21 cells. Further investigation via RNAi or specific inhibitors, or co-immunoprecipitation demonstrated that agonist-induced BmTKRPR internalization was mediated by PKC, GRK5 and ß-arrestin2/BmKurtz. In addition, we also observed that most of the internalized BmTKRP receptors were recycled to the cell surface via early endosomes upon peptide ligand removal. Our study provides the first in-depth information on mechanisms underlying insect TKRP receptor internalization and perhaps aids in the interpretation of the signaling in the regulation of physiological processes.

Niacin Ameliorates Hepatic Steatosis by Inhibiting De Novo Lipogenesis Via a GPR109A-Mediated PKC-ERK1/2-AMPK Signaling Pathway in C57BL/6 Mice Fed a High-Fat Diet.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. Hepatic de novo lipogenesis (DNL) has been suggested to contribute to the pathogenesis of NAFLD. Recent studies have demonstrated that niacin (NA) modulates hepatic DNL through GPR109A. However, the underlying mechanism remains largely unknown. OBJECTIVES: This study aims to elucidate the potential molecular mechanism by which GPR109A inhibits hepatic DNL. METHODS: C57BL/6 wild-type (WT) and Gpr109a knockout (KO) mice (male, 5 wk old) were fed a high-fat diet (60% energy from fat) firstly for 6 wk to generate a diet-induced obese model. Subsequently, they were randomly divided into 4 groups for the next 8-9 wk: WT mice with oral water [WT + vehile (VE)], WT mice with oral NA (50 mM, dissolved in water) (WT + NA), KO mice with oral water (KO + VE), and KO mice with oral NA (50 mM) (KO + NA). Mechanisms were examined in HepG2 cells. Body composition, liver histology, biomarkers of hepatic function, lipid accumulation, and lipid synthesis signals in HepG2 cells were measured. RESULTS: Upon activation, GPR109A apparently protected against obesity and hepatic steatosis (P < 0.05). The concentrations of hepatic Tnf-α in the WT + NA group were about 50% of those in the WT + VE group (P < 0.05). The activities of serum alanine transaminase and aspartate transaminase were 26.7% and 53.5% lower in the WT + NA group than in the WT + VE group, respectively (P < 0.05). In HepG2 cells, activation of GPR109A resulted in remarkable inhibition of oleic acid-induced lipid accumulation via a protein kinase C-extracellular signal-regulated kinase-1/2-AMP-activated protein kinase signaling pathway. CONCLUSIONS: NA inhibits hepatic lipogenesis in C57BL/6 mice through a GPR109A-mediated signaling pathway, consistent with the mechanistic studies in HepG2 cells, suggesting its potential for treatment of NAFLD and other fatty liver diseases.

Pharmacological characterization, cellular localization and expression profile of NPY receptor subtypes Y2 and Y7 in large yellow croaker, Larimichthys crocea.

Neuropeptide Y (NPY) receptors are suggested to mediate the multi-physiological functions of NPY family peptides, such as food intake, in teleost fish. However, the structure and signaling of fish NPY receptors are yet to be fully elucidated. In this study, we report the cloning and characterization of two neuropeptide Y receptor subtypes, Y2 (NPY2R) and Y7 (NPY7R), in yellow croaker Larimichthys crocea (L. crocea) (LcNPY2R, LcNPY7R). The gene structure, pharmacological characterization, cell location, and tissue expression of these two receptors were explored. The phylogenetic results showed that LcNPY2R and LcNPY7R had typical G protein-coupled receptor profiles, associated with the Y2 subfamily, with coding sequences that are highly conserved in vertebrates. The expression of both LcNPY2R and LcNPY7R could be activated by LcNPY in HEK293 cells. However, truncated LcNPY18-36 was only able to activate LcNPY2R at the same level as full length LcNPY. Expression analysis revealed that LcNPY2R mRNA was predominantly expressed in the intestine and liver, whereas LcNPY7R was expressed in the stomach, which indicated that both receptors were related to the digestive system. Overall, our data establishes a molecular basis to determine the actions of LcNPY2R and LcNPY7R, which could be used to elucidate the conserved roles of these receptor-ligand pairs in vertebrates.

Elevenin signaling modulates body color through the tyrosine-mediated cuticle melanism pathway.

Elevenin is a newly discovered novel neuropeptide. Knockdown of either elevenin or orphan receptor NlA42 transcript expression by RNA interference caused severe cuticle melanization in the brown planthopper (BPH). Injection of a synthetic elevenin peptide not only rescued the body color phenotype in dselevenin-pretreated individuals but also suppressed melanization of black insects grown in natural conditions. Real-time quantitative PCR results revealed that elevenin expression levels were highest in the brain and salivary gland. Immunohistochemistry analysis confirmed that a precursor peptide of elevenin was generated in the salivary gland, suggesting that the salivary gland might be an important neurosecretory tissue in addition to the brain in BPH. Furthermore, double-strand RNA-mediated silencing of elevenin and NlA42 resulted in down-regulation of arylalkylamine-N-acetyltransferase and up-regulation of tyrosine hydroxylase, whereas elevenin peptide injection resulted in up-regulation of N-ß-alanyldopamine synthase and aspartate 1-decarboxylase, indicating a complex regulation network for cuticle pigmentation. In addition, functional characterization demonstrated that NlA42 is a cognate receptor for elevenin, and couples to Gq and Gs proteins, triggering both PLC/Ca2+/PKC and AC/cAMP/PKA signaling pathways in response to elevenin treatment. These findings suggest that the elevenin signaling functions control BPH body color through the tyrosine-mediated cuticle melanism pathway.-Wang, S.-L., Wang, W.-W., Ma, Q., Shen, Z.-F., Zhang, M.-Q., Zhou, N.-M., Zhang, C.-X. Elevenin signaling modulates body color through the tyrosine-mediated cuticle melanism pathway.

New Insights in Cannabinoid Receptor Structure and Signaling.

BACKGROUND: Cannabinoid has long been used for medicinal purposes. Cannabinoid signaling has been considered the therapeutic target for treating pain, addiction, obesity, inflammation, and other diseases. Recent studies have suggested that in addition to CB1 and CB2, there are non-CB1 and non-CB2 cannabinoid-related orphan GPCRs including GPR18, GPR55, and GPR119. In addition, CB1 and CB2 display allosteric binding and biased signaling, revealing correlations between biased signaling and functional outcomes. Interestingly, new investigations have indicated that CB1 is functionally present within the mitochondria of striated and heart muscles directly regulating intramitochondrial signaling and respiration. CONCLUSION: In this review, we summarize the recent progress in cannabinoid-related orphan GPCRs, CB1/CB2 structure, Gi/Gs coupling, allosteric ligands and biased signaling, and mitochondria-localized CB1, and discuss the future promise of this research.

Niacin fine-tunes energy homeostasis through canonical GPR109A signaling.

The incidence of overweight and obesity has become a global public health problem, constituting a major risk factor for numerous comorbidities. Despite tremendous efforts, effective pharmacological agents for the treatment of obesity are still limited. Here, we showed that in contrast to lactate receptor GPR81, niacin receptor GPR109A-deficient mice had progressive weight gain and hepatic fat accumulation. Using high-fat diet-induced mouse model of obesity, we demonstrated that niacin treatment apparently protected against obesity without affecting food intake in wild-type mice but not in GPR109A-deficient mice. Further investigation showed that niacin treatment led to a remarkable inhibition of hepatic de novo lipogenesis. Additionally, we demonstrated that niacin treatment triggered brown adipose tissue and/or white adipose tissue thermogenic activity via activation of GPR109A. Moreover, we observed that mice exposed to niacin exhibited a dramatic decrease in intestinal absorption of sterols and fatty acids. Taken together, our findings demonstrate that acting on GPR109A, niacin shows the potential to maintain energy homeostasis through multipathways, representing a potential approach to the treatment of obesity, diabetes and cardiovascular disease.-Ye, L., Cao, Z., Lai, X., Wang, W., Guo, Z., Yan, L., Wang, Y., Shi, Y., Zhou, N. Niacin fine-tunes energy homeostasis through canonical GPR109A signaling.

Effects of Oncogenic Gαq and Gα11 Inhibition by FR900359 in Uveal Melanoma.

Uveal melanoma is the most common intraocular tumor in adults and often metastasizes to the liver, leaving patients with few options. Recurrent activating mutations in the G proteins, Gαq and Gα11, are observed in approximately 93% of all uveal melanomas. Although therapeutic intervention of downstream Gαq/11 targets has been unsuccessful in treating uveal melanoma, we have found that the Gαq/11 inhibitor, FR900359 (FR), effectively inhibits oncogenic Gαq/11 signaling in uveal melanoma cells expressing either mutant Gαq or Gα11. Inhibition of oncogenic Gαq/11 by FR results in cell-cycle arrest and induction of apoptosis. Furthermore, colony formation is prevented by FR treatment of uveal melanoma cells in 3D-cell culture, providing promise for future in vivo studies. This suggests direct inhibition of activating Gαq/11 mutants may be a potential means of treating uveal melanoma. IMPLICATIONS: Oncogenic Gαq/11 inhibition by FR900359 may be a potential treatment option for those with uveal melanoma.