PTTH-Torso Signaling System Controls Developmental Timing, Body Size, and Reproduction through Regulating Ecdysone Homeostasis in the Brown Planthopper, Nilaparvata lugens.

In holometabolous insects, such as Drosophila and Bombyx, prothoracicotropic hormone (PTTH) is well established to be critical in controlling developmental transitions and metamorphosis by stimulating the biosynthesis of ecdysone in the prothoracic glands (PGs). However, the physiological role of PTTH and the receptor Torso in hemimetabolous insects remains largely unexplored. In this study, homozygous PTTH- and Torso-null mutants of the brown planthopper (BPH), Nilaparvata lugens, were successfully generated by employing clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR-Cas9). Further characterization showed that both NlPTTH-/- and NlTorso-/- mutants exhibited prolonged nymphal duration and increased final adult size. Enzyme-linked immunosorbent assay (ELISA) revealed that NlPTTH-/- and NlTorso-/- mutants exhibited a significant reduction in 20-hydroxyecdysone (20E) in fifth-instar nymphs at 48 h post-ecdysis compared to Wt controls. Furthermore, our results indicated that both NlPTTH-/- and NlTorso-/- mutants had shortened lifespan, reduced female fecundity, and reduced egg hatching rates in adults. These findings suggest a conserved role for the PTTH-Torso signaling system in the regulation of developmental transitions by stimulating ecdysone biosynthesis in hemimetabolous insects.

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.

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.

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.

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.

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.

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.

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.

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.

Structural basis for the interaction of diapause hormone with its receptor in the silkworm, Bombyx mori.

Diapause hormone (DH) is a 24-aa amidated neuropeptide that elicits the embryonic diapause of the silkworm, Bombyx mori ( Bommo), via sensitive and selective interaction with its receptor, Bommo DH receptor ( Bommo-DHR). Previous studies of the structure-activity relationship of Bommo-DH were all based on an in vivo diapause-induction bioassay, which has provided little information on the structure of Bommo-DHR or its iteration with DH. Here, to unveil the interaction of Bommo-DH with its receptor, N-terminally truncated analogs and alanine-scanning mutants of Bommo-DH were chemically synthesized and functionally evaluated by using a Cy5.5-labeled Bommo-DH competitive binding assay and Bommo-DHR-based functional assays, including cAMP assay and Ca2+ mobilization assay. Our study demonstrates that the C-terminal residues of Arg23 and Leu24 of Bommo-DH are essential for the binding and activation of Bommo-DHR, and that Trp19 and Phe20 also contribute to the functional activity of Bommo-DH. In contrast, when Gly21 or Pro22 were replaced with alanine, both mutants exhibited binding and signaling activities that were indistinguishable from the wild-type peptide. Furthermore, our homology modeling and molecular dynamics simulations, together with experimental validations, have identified the residues of Glu89, Phe172, Phe194, and Tyr299 in Bommo-DHR that are critically involved in the interaction with Bommo-DH. These results may deepen our understanding of the interactions of class-A GPCRs with their peptidic ligands, particularly those between pheromone biosynthesis-activating neuropeptide/DH family neuropeptides and their cognate receptors.-Shen, Z., Jiang, X., Yan, L., Chen, Y., Wang, W., Shi, Y., Shi, L., Liu, D., Zhou, N. Structural basis for the interaction of diapause hormone with its receptor in the silkworm, Bombyx mori.

Bombyx neuropeptide G protein-coupled receptor A7 is the third cognate receptor for short neuropeptide F from silkworm.

The short neuropeptide F (sNPF) neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects. In the silkworm (Bombyx mori) two orphan G protein-coupled receptors, Bombyx neuropeptide G protein-coupled receptor (BNGR) A10 and A11, have been identified as cognate receptors for sNPFs, but other sNPF receptors and their signaling mechanisms in B. mori remain unknown. Here, we cloned the full-length cDNA of the orphan receptor BNGR-A7 from the brain of B. mori larvae and identified it as a receptor for Bombyx sNPFs. Further characterization of signaling and internalization indicated that BNGR-A7, -A10, and -A11 are activated by direct interaction with synthetic Bombyx sNPF-1 and -3 peptides. This activation inhibited forskolin or adipokinetic hormone-induced adenylyl cyclase activity and intracellular Ca2+ mobilization via a Gi/o-dependent pathway. Upon activation by sNPFs, BNGR-A7, -A10, and -A11 evoked ERK1/2 phosphorylation and underwent internalization. On the basis of these findings, we designated the receptors BNGR-A7, -A10, and -A11 as Bommo-sNPFR-1, -2, and -3, respectively. Moreover, the results obtained with quantitative RT-PCR analysis revealed that the three Bombyx sNPF receptor subtypes exhibit differential spatial and temporal expression patterns, suggesting possible roles of sNPF signaling in the regulation of a wide range of biological processes. Our findings provide the first in-depth information on sNPF signaling for further elucidation of the roles of the Bombyx sNPF/sNPFR system in the regulation of physiological activities.

BNGR-A25L and -A27 are two functional G protein-coupled receptors for CAPA periviscerokinin neuropeptides in the silkworm Bombyx mori.

CAPA peptides, such as periviscerokinin (PVK), are insect neuropeptides involved in many signaling pathways controlling, for example, metabolism, behavior, and reproduction. They are present in a large number of insects and, together with their cognate receptors, are important for research into approaches for improving insect control. However, the CAPA receptors in the silkworm (Bombyx mori) insect model are unknown. Here, we cloned cDNAs of two putative CAPA peptide receptor genes, BNGR-A27 and -A25, from the brain of B. mori larvae. We found that the predicted BNGR-A27 ORF encodes 450 amino acids and that one BNGR-A25 splice variant encodes a full-length isoform (BNGR-A25L) of 418 amino acid residues and another a short isoform (BNGR-A25S) of 341 amino acids with a truncated C-terminal tail. Functional assays indicated that both BNGR-A25L and -A27 are activated by the PVK neuropeptides Bom-CAPA-PVK-1 and -PVK-2, leading to a significant increase in cAMP-response element-controlled luciferase activity and Ca2+ mobilization in a Gq inhibitor-sensitive manner. In contrast, BNGR-A25S was not significantly activated in response to the PVK peptides. Moreover, Bom-CAPA-PVK-1 directly bound to BNGR-A25L and -A27, but not BNGR-A25S. Of note, CAPA-PVK-mediated ERK1/2 phosphorylation and receptor internalization confirmed that BNGR-A25L and -A27 are two canonical receptors for Bombyx CAPA-PVKs. However, BNGR-A25S alone is a nonfunctional receptor but serves as a dominant-negative protein for BNGR-A25L. These results provide evidence that BNGR-A25L and -A27 are two functional Gq-coupled receptors for Bombyx CAPA-PVKs, enabling the further elucidation of the endocrinological roles of Bom-CAPA-PVKs and their receptors in insect biology.

Molecular and functional characterization of tumor-induced factor (TIF): Hamster homolog of CXCL3 (GROγ) displays tumor suppressive activity.

Previously our lab has created a mouse ovarian xenograft model of copy number variation (CNV)-mediated G protein-coupled receptor (GPCR) MAS-driven tumorigenesis, and RNA profiling identified a putative chemokine tumor-induced factor (Tif). Sequence analysis and chemotactic study suggested that Tif was likely to be a hamster homolog of human GROγ (CXCL3) [IJC 125 (2009): 1316-1327]. In the present study, we report the molecular and functional characterization of the Tif gene. Genomic study of CHO-K1 cells indicated that Tif gene consisted of 4 exons, characterized with an antisense B1 element which is embedded in the fourth exon. Two Tif transcripts were identified which shared identical sequences except that a string of 71-nt derived from the antisense B1 element was deficient in the shorter transcript. Of interests, B1-like RNA ladder was detected in xenografts. Functional studies showed that TIF induced chemotaxis and neovessel formation. Pharmacological studies suggested that TIF activated Gi-coupled CXCR2 and induced both calcium mobilization and ERK1/2 phosphorylation, and suppressed forskolin-stimulated cAMP accumulation. In addition, secreted matured TIF functioned as an autocrine factor and promoted anchorage-independent growth. Unexpectedly, TIF delayed the onset of tumor formation, possibly via suppressing proliferation of stromal fibroblasts. However, TIF did not exert any inhibitory effect on tumor growth. Potentially, TIF could be used for preventing cancer relapse.

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.

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.

Agonist-Activated Bombyx Corazonin Receptor Is Internalized via an Arrestin-Dependent and Clathrin-Independent Pathway.

Agonist-induced internalization plays a key role in the tight regulation of the extent and duration of G protein-coupled receptor signaling. Previously, we have shown that the Bombyx corazonin receptor (BmCrzR) activates both Gαq- and Gαs-dependent signaling cascades. However, the molecular mechanisms involved in the regulation of the internalization and desensitization of BmCrzR remain to be elucidated. Here, vectors for expressing BmCrzR fused with enhanced green fluorescent protein (EGFP) at the C-terminal end were used to further characterize BmCrzR internalization. We found that the BmCrzR heterologously expressed in HEK-293 and BmN cells was rapidly internalized from the plasma membrane into the cytoplasm in a concentration- and time-dependent manner via a ß-arrestin (Kurtz)-dependent and clathrin-independent pathway in response to agonist challenge. While most of the internalized receptors were recycled to the cell surface via early endosomes, some others were transported to lysosomes for degradation. Assays using RNA interference revealed that both GRK2 and GRK5 were essentially involved in the regulation of BmCrzR phosphorylation and internalization. Further investigations indicated that the identified cluster of Ser/Thr residues ((411)TSS(413)) was responsible for GRK-mediated phosphorylation and internalization. This is the first detailed investigation of the internalization and trafficking of Bombyx corazonin receptors.

Agonist-induced activation of histamine H3 receptor signals to extracellular signal-regulated kinases 1 and 2 through PKC-, PLD-, and EGFR-dependent mechanisms.

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 ßγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, ß-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.

Bombyx mori prothoracicostatic peptide receptor is allosterically activated via a Gα(i/o)-protein-biased signalling cascade by Drosophila sex peptide.

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 Ca²âº 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 Ca²âº mobilization and arrestin recruitment.

Melatonin receptor type 1 signals to extracellular signal-regulated kinase 1 and 2 via Gi and Gs dually coupled pathways in HEK-293 cells.

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 Gßγ 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.

Activation of BNGR-A24 by direct interaction with tachykinin-related peptides from the silkworm Bombyx mori leads to the Gq- and Gs-coupled signaling cascades.

Tachykinins constitute one of the largest peptide families in the animal kingdom and exert their diverse actions via G protein-coupled receptors (GPCRs). In this study, the Bombyx tachykinin-related peptides (TKRPs) were identified as specific endogenous ligands for the Bombyx neuropeptide GPCR A24 (BNGR-A24) and thus designated BNGR-A24 as BmTKRPR. Using both mammalian cell line HEK293 and insect cell line Sf21, further characterization demonstrated that BmTKRPR was activated, thus resulting in intracellular accumulation of cAMP, Ca(2+) mobilization, and ERK1/2 phosphorylation in a Gs and Gq inhibitor-sensitive manner. Moreover, quantitative reverse transcriptase polymerase chain reaction analysis and dsRNA-mediated knockdown experiments suggested a possible role for BmTKRPR in the regulation of feeding and growth. Our findings enhance the understanding of the Bombyx TKRP system in the regulation of fundamental physiological processes.

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.

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.