Bombyx mori epidermal growth factor receptor is required for nucleopolyhedrovirus replication.

Baculovirus-host interactions are important models for studying the biological control of lepidopteran pests. Research on baculovirus-host interactions has focussed on baculovirus manipulation of cellular signalling pathways, including the extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinases/protein kinase B (PI3K/Akt) signalling pathways. However, the mechanism underlying ERK and PI3K/Akt activation and function in response to baculovirus infection remains poorly understood. Here, we demonstrated that baculovirus activated the Bombyx mori ERK and PI3K/Akt signalling pathways via the B. mori epidermal growth factor receptor (BmEGFR). To further characterize the function of the BmEGFR/ERK signalling pathway in baculovirus replication, we calculated genome-wide changes in kinase-chromatin interactions for ERK after baculovirus infection using chromatin immunoprecipitation followed by high-throughput sequencing. A Gene Ontology analysis showed that virus infection had effects on the biological regulation, cellular process and metabolic process pathways. Moreover, ERK was shown to regulate the transcription of late viral genes. Taken together, our results suggest that baculoviruses manipulate components of the host cell machinery for replication via modulation of the BmEGFR signalling pathway.

Chemokine (C-X-C motif) ligand 1 is a myokine induced by palmitate and is required for myogenesis in mouse satellite cells.

AIM: The functional significance of the myokines, cytokines and peptides produced and released by muscle cells has not been fully elucidated. The purpose of this study was to identify a myokine with increased secretion levels in muscle cells due to saturated fatty acids and to examine the role of the identified myokine in the regulation of myogenesis. METHODS: Human primary myotubes and mouse C2C12 myotubes were used to identify the myokine; its secretion was stimulated by palmitate loading. The role of the identified myokine in the regulation of the activation, proliferation, differentiation and self-renewal was examined in mouse satellite cells (skeletal muscle stem cells). RESULTS: Palmitate loading promoted the secretion of chemokine (C-X-C motif) ligand 1 (CXCL1) in human primary myotubes, and it also increased CXCL1 gene expression level in C2C12 myotubes in a dose- and time-dependent manner. Palmitate loading increased the production of reactive oxygen species along with the activation of nuclear factor-kappa B (NF-κB) signalling. Pharmacological inhibition of NF-κB signalling attenuated the increase in CXCL1 gene expression induced by palmitate and hydrogen peroxide. Palmitate loading significantly increased CXC receptor 2 gene expression in undifferentiated cells. CXCL1 knockdown attenuated proliferation and myotube formation by satellite cells, with reduced self-renewal. CXCL1 knockdown also significantly decreased the Notch intracellular domain protein level. CONCLUSION: These results suggest that secretion of the myokine CXCL1 is stimulated by saturated fatty acids and that CXCL1 promotes myogenesis from satellite cells to maintain skeletal muscle homeostasis.

Characterization and evolutionary analysis of tributyltin-binding protein and pufferfish saxitoxin and tetrodotoxin-binding protein genes in toxic and nontoxic pufferfishes.

Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long-standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin-binding protein type 2 (TBT-bp2) domains. In this study, we examined the evolutionary dynamics of TBT-bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR-based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT-bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT-bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT-bp2 genes. In addition, a detailed comparison of Takifugu TBT-bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT-bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes.

Leptin improves fatty liver independently of insulin sensitization and appetite suppression in hepatocyte-specific Pten-deficient mice with insulin hypersensitivity.

Nonalcoholic fatty liver disease (NAFLD) is recognized as the hepatic component of the metabolic syndrome. Although NAFLD is a major cause of cirrhosis and cancer of the liver of unknown cause, no established pharmacological treatment for NAFLD has been established yet. It has been reported that leptin treatment improved fatty liver dramatically as well as insulin resistance and hyperphagia in patients with lipodystrophy. However, it is unclear whether leptin improves fatty liver independently of these metabolic improvements. We investigated the liver effect of leptin independently of insulin sensitization and appetite suppression using hepatocyte-specific Pten-deficient (AlbCrePtenff) mouse, a model of severe fatty liver with insulin hypersensitivity. Male AlbCrePtenff mice were infused subcutaneously with leptin (20 ng/g/h) for 2 weeks using osmotic minipumps. Leptin infusion effectively reduced liver weight, liver triglyceride content, and glutamate pyruvate transaminase (GPT) concentrations as well as food intake and body weight without the change of plasma insulin concentration in AlbCrePtenff mice. Pair-feeding also reduced body weight but not liver triglyceride content. Pair feeding reduced α1 and α2 AMP-activated protein kinase (AMPK) activities and PGC1α gene expression in the liver, while leptin infusion unchanged them. The present study clearly demonstrated that leptin improve fatty liver independently of insulin sensitization and suppression of food intake. It was suggested that leptin improves fatty liver by stimulation of ß-oxidation in the liver. The present study might provide a further understanding on the mechanism of metabolic effect of leptin.

Middle region of FancM interacts with Mhf and Rmi1 in silkworms, a species lacking the Fanconi anaemia (FA) core complex.

The Fanconi anaemia (FA) pathway is responsible for interstrand crosslink (ICL) repair. Among the FA core complex components, FANCM is believed to act as a damage sensor for the ICL-blocked replication fork and also as a molecular platform for FA core complex assembly and interaction with Bloom's syndrome (BS) complex that is thought to play an important role in the processing of DNA structures such as stalled replication forks. In the present study, we found that in silkworms, Bombyx mori, a species lacking the major FA core complex components (FANCA, B, C, E, F, and G), FancM is required for FancD2 monoubiquitination and cell proliferation in the presence of mitomycin C (MMC). Silkworm FancM (BmFancM) was phosphorylated in the middle regions, and the modification was associated with its subcellular localization. In addition, BmFancM interacted with Mhf1, a histone-fold protein, and Rmi1, a subunit of the BS complex, in the different regions. The interaction region containing at least these two protein-binding domains played an essential role in FancM-dependent resistance to MMC. Our results suggest that BmFancM also acts as a platform for recruitment of both the FA protein and the BS protein, although the silkworm genome seems to lose FAAP24, a FancM-binding partner protein in mammals.

A MC motif in silkworm Argonaute 1 is indispensible for translation repression.

Small RNA-mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA-induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double-stranded RNA (dsRNA)-induced RNAi, whereas Ago3 and Siwi are involved in the Piwi-interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)-mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA-decapping enzyme 2 (Dcp2) protein in the Drosophila processing-bodies (P-bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P-bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori.

PhiC31 integrase-mediated cassette exchange in silkworm embryos.

To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4-6 h after oviposition and recovered plasmid DNAs from the embryos 3 days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.

Impairment of fear-conditioning responses and changes of brain neurotrophic factors in diet-induced obese mice.

Recent epidemiological studies demonstrate that obesity is related to a high incidence of cognitive impairment. In the present study, cognitive behaviours in diet-induced obese (DIO) mice fed 60% high-fat diet for 16 weeks were compared with those in mice fed a control diet (CD) in fear-conditioning tests including both contextual and cued elements that preferentially depend on the hippocampus and amygdala, respectively. Furthermore, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) content in the brain areas was examined in both CD and DIO mice. In fear-conditioning tests, the freezing percentages of both contextual fear and cued fear responses in DIO mice were significantly lower than in CD mice. BDNF content in the cerebral cortex and hippocampus of DIO mice was significantly lower than that in CD mice. Its receptor, full-length TrkB, in the amygdala of DIO mice was significantly decreased compared to that in CD mice, although not in the cerebral cortex, hippocampus and hypothalamus. By contrast, NT-3 content in the hippocampus, amygdala and hypothalamus of DIO mice was significantly higher than that in CD mice. Its receptor, full-length TrkC, was not significantly different between CD and DIO mice. The present study demonstrates that DIO mice show impairment of both hippocampus-dependent contextual and amygdala-dependent cued responses in the fear-conditioning tests, as well as an imbalance in the interaction between the BDNF and NT-3 systems in the cerebral cortex, hippocampus and amygdala related to cognition and fear.

Dynamic changes in the dural sac cross-sectional area on axial loaded MR imaging: is there a difference between degenerative spondylolisthesis and spinal stenosis?

BACKGROUND AND PURPOSE: Axial loaded MR imaging, which can simulate the spinal canal of patients in a standing position, demonstrates a significant reduction of the DCSA compared with conventional MR imaging and provides valuable imaging findings in the assessment of the lumbar spinal canal. The purpose of this study was to compare the DCSA on axial loaded MR imaging between patients with DS and SpS. MATERIALS AND METHODS: Eighty-eight consecutive patients were divided into DS and SpS groups. DCSA on conventional MR imaging and axial loaded MR imaging and changes in the DCSA induced by axial loading were compared between DS and SpS groups. The prevalence of a significant change (>15 mm(2)) in the DCSA was compared between the 2 groups. RESULTS: Axial loaded MR imaging demonstrated significantly smaller DCSA in the DS group (35 ± 22 mm(2)) than in the SpS group (50 ± 31 mm(2)), though conventional MR imaging did not show any differences between the 2 groups. The change in the DCSA induced by axial loading was significantly larger in the DS group (17 ± 12 mm(2)) compared with the SpS group (8 ± 8 mm(2)). The prevalence of a >15-mm(2) change in the DCSA was significantly higher in the DS group (62.5%) than in the SpS group (16.7%) (odds ratio, 8.33; 95% confidence interval, 3.09-22.50). CONCLUSIONS: Axial loaded MR imaging demonstrated significantly larger changes in the DCSA in patients with DS compared those with SpS. A significant change in the DCSA was more frequently observed in patients with DS. Axial loaded MR imaging may therefore be a more useful tool to decrease the risk of underestimating the spinal canal narrowing in patients with DS than in those with SpS.

Feasibility and efficiency of indirect lifestyle interventions in offspring of type 2 diabetic patients.

INTRODUCTION: Individuals genetically predisposed to type 2 diabetes represent an important target for preventive strategies. Genetic screening, based on information about individual genetic variants, will be possible technically, but translational research in this field is still insufficient. Family history thus represents a useful tool for detecting genetically high-risk populations in this post-genomic era. OBJECTIVES: The purpose of this pilot study was to investigate the feasibility and efficiency of indirect lifestyle interventions in offspring of type 2 diabetic patients. METHODS: Offspring were recruited from 74 diabetic (Group 1) and 39 non-diabetic (Group 2, control group) patients. A lifestyle intervention was conducted by mail, a total of 3 times, every 3 months. Lifestyle related to diet and physical activity was assessed using a self-administered questionnaire. RESULTS: Ten offspring of type 2 diabetic and 6 of non-diabetic patients participated in this study. Total energy intake decreased after 3 interventions in both of the groups (Group 1: 305 ± 228.8 kcal/day, p = 0.004; Group 2: 82 ± 65.6 kcal/day, p = 0.04); however, the effect of intervention was significantly greater in Group 1 compared to Group 2 (p = 0.021). Physical activity and other physical outcomes were stable in normal levels during the study period in both of the groups. CONCLUSIONS: The intervention program helped to reduce total energy intake in offspring of type 2 diabetic patients more than in the control group, but the acceptance rate of the intervention program was disappointingly low. Further consideration is required to access and motivate offspring to develop precautionary lifestyle principles.

Irbesartan treatment up-regulates hepatic expression of PPARalpha and its target genes in obese Koletsky (fa(k)/fa(k)) rats: a link to amelioration of hypertriglyceridaemia.

BACKGROUND AND PURPOSE: Hypertriglyceridaemia is associated with an increased risk of cardiovascular disease. Irbesartan, a well-established angiotensin II type 1 receptor (AT(1)) blocker, improves hypertriglyceridaemia in rodents and humans but the underlying mechanism of action is unclear. EXPERIMENTAL APPROACH: Male obese Koletsky (fa(k)/fa(k)) rats, which exhibit spontaneous hypertension and metabolic abnormalities, received irbesartan (40 mg x kg(-1) x day(-1)) or vehicle by oral gavage over 7 weeks. Adipocyte-derived hormones in plasma were measured by ELISA. Gene expression in liver and other tissues was assessed by real-time PCR and Western immunoblotting. KEY RESULTS: In Koletsky (fa(k)/fa(k)) rats irbesartan lowered plasma concentrations of triglycerides and non-esterified fatty acids, and decreased plasma insulin concentrations and the homeostasis model assessment of insulin resistance index. However, this treatment did not affect food intake, body weight, epididymal white adipose tissue weight, adipocyte size and plasma leptin concentrations, although plasma adiponectin was decreased. Irbesartan up-regulated hepatic expression of mRNAs corresponding to peroxisome proliferator-activated receptor (PPAR)alpha and its target genes (carnitine palmitoyltransferase-1a, acyl-CoA oxidase and fatty acid translocase/CD36) that mediate hepatic fatty acid uptake and oxidation; the increase in hepatic PPARalpha expression was confirmed at the protein level. In contrast, irbesartan did not affect expression of adipose PPARgamma and its downstream genes or hepatic genes that mediate fatty acid synthesis. CONCLUSIONS AND IMPLICATIONS: These findings demonstrate that irbesartan treatment up-regulates PPARalpha and several target genes in liver of obese spontaneously hypertensive Koletsky (fa(k)/fa(k)) rats and offers a novel insight into the lipid-lowering mechanism of irbesartan.

Angiotensin II type 1 receptor-independent beneficial effects of telmisartan on dietary-induced obesity, insulin resistance and fatty liver in mice.

AIMS/HYPOTHESIS: Evidence suggests that telmisartan, an angiotensin II type 1 receptor (AT1) blocker and peroxisome proliferator-activated receptor-gamma partial agonist, has beneficial actions that limit development of the metabolic syndrome and diabetes. However, the role played by AT1 inhibition in metabolic effects elicited by telmisartan remains uncertain. Here we isolated the metabolic effects of telmisartan from AT1 antagonism. METHODS: Male At1a (also known as Agtr1a)-deficient mice were fed a standard diet or 60% high-fat diet; those on high-fat diet were co-administered telmisartan (3 mg kg(-1) day(-1) by oral gavage) or vehicle for 12 weeks. RESULTS: In At1a-null mice, telmisartan prevented high-fat-diet-induced increases in (1) body weight, epididymal and inguinal white adipose tissue weight, adipocyte size and plasma leptin concentration; (2) plasma glucose and insulin concentrations and HOMA index; and (3) liver weight and triacylglycerol content. Insulin tolerance testing also indicated that telmisartan improved the high-fat-diet-induced reduction of glucose-lowering by insulin. CONCLUSIONS/INTERPRETATION: The present findings demonstrate beneficial, AT1-independent effects of the AT1 blocker telmisartan on dietary-induced obesity, insulin resistance and fatty liver in animals.

An adipose tissue-independent insulin-sensitizing action of telmisartan: a study in lipodystrophic mice.

Adipose tissue plays an important role in energy balance and metabolism and is the major target for insulin-sensitizing peroxisome proliferator-activated receptor (PPAR) gamma agonists. The angiotensin II type 1 receptor blocker telmisartan, a partial agonist of PPAR-gamma, has been demonstrated to improve insulin sensitivity. However, there is uncertainty about the sites of its action. Here, we demonstrate that treatment with telmisartan (3 mg/kg p.o.) for 7 weeks decreased plasma glucose levels in oral glucose and insulin tolerance tests and the index of the homeostasis model assessment of insulin resistance in A-ZIP/F-1 transgenic mice, an animal model of lipodystrophy. These effects were accompanied by decreases in circulating triglyceride and fatty acid levels. However, this treatment did not affect body weight and plasma adiponectin, leptin, and corticosterone levels. In A-ZIP/F-1 mouse liver the transcripts encoding PPAR-gamma and its downstream lipogenic genes were highly up-regulated, consistent with increased hepatic triglyceride content and lipid droplet accumulation. Telmisartan reversed these effects and also down-regulated mRNAs encoding gluconeogenic genes. Thus, the present findings are consistent with a novel mode of insulin-sensitizing action of telmisartan, involving an adipose tissue-independent pathway. Telmisartan-elicited down-regulation of hepatic expression of PPAR-gamma-regulated lipogenic genes is associated with amelioration of fatty liver.

Facet cyst haematoma in the lumbar spine: a report of four cases.

We present 4 cases of facet cyst haematoma in the ligamentum flavum of the lumbar spine. All patients presented with a one-to-3-month history of back pain or numbness in the legs, and sudden neurological deterioration. One also developed cauda equina syndrome and another developed radiculopathy. In all cases, magnetic resonance imaging showed a mass with high signal intensity on both T1- and T2-weighted images. Facet arthrography and computed tomography revealed communication between the mass and the neighbouring facet joint. The haematomas were removed en bloc with the ligamentum flavum. They were surrounded by the ligament and contained degenerated and lacerated elastic fibres but no synovial lining cells. Facet cyst haematoma is so-named because of bleeding from tissue adjacent to the facet joint into a pre-existing facet cyst.

Overexpression of E2F-5 correlates with a pathological basal phenotype and a worse clinical outcome.

The purpose of the present study is to identify genes that contribute to cell proliferation or differentiation of breast cancers independent of signalling through the oestrogen receptor (ER) or human epidermal growth factor receptor 2 (HER2). An oligonucleotide microarray assayed 40 tumour samples from ER(+)/HER2(-), ER(+)/HER2(+), ER(-)/HER2(+), and ER(-)/HER2(-) breast cancer tissues. Quantitative reverse transcriptase PCR detected overexpression of a cell cycle-related transcription factor, E2F-5, in ER-negative breast cancers, and fluorescence in situ hybridisation detected gene amplification of E2F-5 in 5 out of 57 (8.8%) breast cancer samples. No point mutations were found in the DNA-binding or DNA-dimerisation domain of E2F-5. Immunohistochemically, E2F-5-positive cancers correlated with a higher Ki-67 labelling index (59.5%, P=0.001) and higher histological grades (P=0.049). E2F-5-positive cancers were found more frequently in ER(-)/progesterone receptor (PgR)(-)/HER2(-) cancer samples (51.9%, P=0.0049) and in breast cancer samples exhibiting a basal phenotype (56.0%, P=0.0012). Disease-free survival in node-negative patients with E2F-5-positive cancers was shorter than for patients with E2F-5-negative cancers. In conclusion, we identify, for the first time, a population of breast cancer cells that overexpress the cell cycle-related transcription factor, E2F-5. This E2F-5-positive breast cancer subtype was associated with an ER(-)/PgR(-)/HER2(-) status, a basal phenotype, and a worse clinical outcome.

Beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and a high-fat diet.

AIMS/HYPOTHESIS: We have previously demonstrated the therapeutic usefulness of leptin in lipoatrophic diabetes and insulin-deficient diabetes in mouse models and could also demonstrate its dramatic effects on lipoatrophic diabetes in humans. The aim of the present study was to explore the therapeutic usefulness of leptin in a mouse model of type 2 diabetes with increased adiposity. METHODS: To generate a mouse model mimicking human type 2 diabetes with increased adiposity, we used a combination of low-dose streptozotocin (STZ, 120 microg/g body weight) and high-fat diet (HFD, 45% of energy as fat). Recombinant mouse leptin was infused chronically (20 ng [g body weight](-1) h(-1)) for 14 days using a mini-osmotic pump. The effects of leptin on food intake, body weight, metabolic variables, tissue triacylglycerol content and AMP-activated protein kinase (AMPK) activity were examined. RESULTS: Low-dose STZ injection led to a substantial reduction of plasma insulin levels and hyperglycaemia. Subsequent HFD feeding increased adiposity and induced insulin resistance and further augmentation of hyperglycaemia. In this model mouse mimicking human type 2 diabetes (STZ/HFD), continuous leptin infusion reduced food intake and body weight and improved glucose and lipid metabolism with enhancement of insulin sensitivity. Leptin also decreased liver and skeletal muscle triacylglycerol content accompanied by an increase of alpha2 AMPK activity in skeletal muscle. Pair-feeding experiments demonstrated that leptin improved glucose and lipid metabolism independently of the food intake reduction. CONCLUSIONS/INTERPRETATION: This study demonstrates the beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity, indicating the possible clinical usefulness of leptin as a new glucose-lowering drug in humans.

Seasonal and spatial variations in characteristics of Lake Biwa dissolved organic matter: sorption of pyrene and its derivatives and fluorescence properties.

The objectives of this research were to investigate seasonal and spatial variations in (1) sorption of pyrene and its derivatives onto dissolved organic matter (DOM) and (2) fluorescence properties of DOM in Lake Biwa, Japan. In the case of pyrene, sorption coefficient (Kdoc) of Lake Biwa DOM seasonally changed from 1,200 to 3,800 L/kgC. Vertical distribution of Kdoc was affected by thermocline formation in summer, while it was uniformly distributed as a result of vertical mixing in winter. Functional groups affected sorption of pyrene onto Lake Biwa DOM in different manner from that onto Suwannee River fulvic acid. Three-dimensional excitation emission matrices (3D-EEMs) fluorescence spectroscopy was applied to characterize Lake Biwa DOMs and indicated the existence of at least two fluorophores. The two major peaks at Ex230/Em300 and Ex230/Em425 originated from protein-like and fulvic/humic-like substances, respectively. The peak at Ex230/Em300 showed the maximum fluorescence intensity at a depth of 5 m and could be affected by stratification of the water column in summer. On the other hand, the peak at Ex230/Em425 showed similar profiles both in summer and in winter. These results demonstrably showed that sorption of micropollutants and fluorescence properties of Lake Biwa DOMs were seasonally and spatially varied.

Functions of a GnRH receptor heterodimer of the ascidian, Ciona intestinalis.

Gonadotropin-releasing hormone (GnRH) is a ten-amino acid peptide hormone that plays pivotal roles in reproduction in vertebrates and octopus. Recently, six GnRH forms (t-GnRH-3-8) and four GnRH receptor subtypes (Ci-GnRHR-1-4) were identified in the protochordate, Ciona intestinalis. In this study, we show the functional modulation of Ci-GnRHR-1 via heterodimerization with the orphan receptor subtype, Ci-GnRHR-4. The dimerization between Ci-GnRHR-1 and R-4 was detected by co-immunoprecipitation and immunoblot analysis. Binding assays confirmed the binding of t-GnRHs to Ci-GnRHR-1 but not to R-4, and verified no alternation in ligand-binding affinity between Ci-GnRHR-1 homodimer and Ci-GnRHRI&4 heterodimer. The heterodimer was found to stimulate the elevation of intracellular calcium, time-extension of ERK phosphorylation, and up-regulation of cell proliferation, all in a ligand specific manner, compared with the Ci-GnRHR-1 homodimer. In combination, these results indicated that Ci-GnRHR-4 is not an inactive receptor, but a modulatory factor for Ci-GnRHR-1 in C. intestinalis.