Structure-activity relationship study on small peptidic GPR54 agonists

Metastin (kisspeptin-54) is an endogenous ligand that modulates gonadotropin-releasing hormone (GnRH) secretion through the interaction with a G protein-coupled receptor (GPCR), GPR54. The short-chain C-terminal decapeptide amide, metastin (45-54) (kisspeptin-10), exerts the identical bioactivities to metastin, such as metastasis suppression of cancer cells and inhibition of trophoblast migration and invasion. In order to understand the structural requirement for GPR54 agonistic activity, structure-activity relationship (SAR) study on pentapeptide-based C-terminal metastin analogues was carried out. As a result, H-Amb-Nal(2)-Gly-Leu-Arg-Trp-NH2 34 was identified as a novel GPR54 agonist that possessed the most potent GPR54 agonistic activity reported so far.     

Establishment and clinical application of enzyme immunoassays for determination of luteinizing hormone releasing hormone and metastin.

Metastin, a 54-residue peptide, was identified as the cognate ligand of human G-protein-coupled receptor GPR54. Since metastin is a gene product of the human metastasis suppressor gene 'KiSS-1', early studies on metastin were focused on its activity as a tumor metastasis suppressor. Recently, there have been some reports that metastin is found in human plasma and is particularly abundant in the plasma of pregnant women. Dysfunction of the GPR54 receptor causes diseases that are characterized by an insufficient release of gonadotropin and lack or delay of pubertal maturation. This information strongly suggests that metastin is involved in the regulation of reproductive endocrine functions. In order to determine the plasma levels of metastin and luteinizing hormone releasing hormone (LHRH) in an isolated hypogonadotropic hypogonadism (IHH) patient, who received intermittent administrations of LHRH, we tried to establish a sensitive and specific enzyme immunoassay. The plasma LHRH levels of the patient were very high, while plasma metastin levels were at almost the same levels as circadian rhythms of healthy male humans. In the central nervous system, metastin stimulates the neuroendocrine reproductive axis. However, the effects of peripheral metastin are not known. Our result suggested that peripheral metastin had a genesis and activity different from central metastin.     

2-Acylamino-4,6-diphenylpyridine derivatives as novel GPR54 antagonists with good brain exposure and in vivo efficacy for plasma LH level in male rats

GPR54 is a G protein-coupled receptor (GPCR) which was formerly an orphan receptor. Recent functional study of GPR54 revealed that the receptor plays an essential role to modulate sex-hormones including GnRH. Thus, antagonists of GPR54 are expected to be novel drugs for sex-hormone dependent diseases such as prostate cancer or endometriosis. We recently reported 2-acylamino-4,6-diphenylpyridines as the first small molecule GPR54 antagonists with high potency. However, the representative compound 1 showed low brain exposure, where GPR54 acts as a modulator of gonadotropins by binding with its endogenous ligand, metastin. In order to discover compounds that have not only potent GPR54 antagonistic activity but also good brain permeability, we focused on converting the primary amine on the side chain to a secondary or tertiary amine, and finally we identified 15a containing a piperazine group. This compound exhibited high affinity to human and rat GPR54, apparent antagonistic activity, and high brain exposure. In addition, intravenous administration of 15a to castrated male rat suppressed plasma LH level, which indicates the possibility of a small molecule GPR54 antagonist as a novel drug for sex-hormone dependent diseases.     

Physical association of GPR54 C-terminal with protein phosphatase 2A

KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.     

Metastin suppresses the motility and growth of CHO cells transfected with its receptor

We recently reported having identified of the ligand for an orphan G-protein-coupled receptor, hOT7T175, as the gene product (68-121)-amide of the metastasis suppressor gene KiSS-1. We further showed that the ligand, which we named "metastin," inhibits chemotaxis and invasion of Chinese hamster ovary (CHO) cells transfected with hOT7T175 cDNA (CHO/h175) in vitro, and pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. In the present study, we investigated the activity of metastin in CHO/h175 cells in greater detail. Metastin significantly suppressed motility in a chemotaxis assay and wound healing assay at 10-100 nM order concentrations. Two N-terminally truncated peptides, metastin(40-54) and metastin(45-54) inhibited the migration of CHO/h175 cells as potently as metastin itself. Metastin also inhibited the spreading, monolayer growth and colony formation in agar (0.8%) of CHO/h175 cells at 10-100 nM concentrations. These results indicate that metastin is a potent inhibitor of cell motility, leading to suppression of cell growth and antimetastatic activity, and suggest that low molecular chemical compounds could replace its activity as a novel antimetastatic agent.     

Elevated Expression of KiSS-1 in Placenta of Chinese Women with Early-Onset Preeclampsia

Preeclampsia (PE) is a heterogeneous syndrome affecting 2% to 8% of all pregnancies and is the world’s leading cause of fetal and maternal morbidity and mortality. In many cases of PE, shallow trophoblast invasion results in inappropriate maternal spiral artery remodeling and impaired placental function. Multiple genes have been implicated in trophoblast invasion, among which are KiSS-1 and GPR54. The gene product of KiSS-1 is metastin, which is a ligand for the receptor GPR54. Both metastin and GPR54 are expressed in the placenta of normal pregnancy and have been implicated in modulating trophoblast invasion through inhibiting migration of trophoblast cells. We have previously reported that the expression level of KiSS-1 was higher in trophoblasts from women with preeclampsia as compared to normal controls. Here, using quantitative RT-PCR, Western blot analysis and immunohistochemistry, we extend our analysis to demonstrate that elevated KiSS-1 expression occurs only in early-onset preeclampsia (ePE) and not late-onset preeclampsia (lPE). However, no difference in the expression levels of GPR54 is observed between ePE, lPE, and normal controls. Further, we show that KiSS-1 expression is also increased in placenta of intrauterine death and birth asphyxia in comparison to normal newborns of ePE and lPE. Our findings suggest that aberrant upregulation of KiSS-1 expression may contribute to the underlying mechanism of ePE as well as birth asphyxia.     

Molecular cloning of the bullfrog kisspeptin receptor GPR54 with high sensitivity to Xenopus kisspeptin

Kisspeptin and its receptor, GPR54, play important roles in mammalian reproduction and cancer development. However, little is known about their function in nonmammalian species. In the present study, we have isolated the cDNA encoding the kisspeptin receptor, GPR54, from the bullfrog, Rana catesbeiana. The bullfrog GPR54 (bfGPR54) cDNA encodes a 379-amino acid heptahelical G protein-coupled receptor. bfGPR54 exhibits 45-46% amino acid identity with mammalian GPR54s and 70-74% identity with fish GPR54s. RT-PCR analysis showed that bfGPR54 mRNA is highly expressed in the forebrain, hypothalamus and pituitary. Upon stimulation by synthetic human kisspeptin-10 with Phe-amide residue at the C-terminus (h-Kiss-10F), bfGPR54 induces SRE-luc activity, a PKC-specific reporter, evidencing the PKC-linked signaling pathway of bfGPR54. Using a blast search, we found a gene encoding a kisspeptin-like peptide in Xenopus. The C-terminal decapeptide of Xenopus kisspeptin shows higher amino acid sequence identity to fish Kiss-10s than mammalian Kiss-10s. A synthetic Xenopus kisspeptin peptide (x-Kiss-12Y) showed a higher potency than mammalian Kiss-10s in the activation of bfGPR54. This study expands our understanding of the physiological roles and molecular evolution of kisspeptins and their receptors.     

The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system

GPR54 is a G-protein-coupled receptor that displays a high percentage of identity in the transmembrane domains with the galanin receptors. The ligand for GPR54 has been identified as a peptide derived from the KiSS-1 gene. KiSS-1 has been shown to have anti-metastatic effects, suggesting that KiSS-1 or its receptor represents a potential therapeutic target. To further our understanding of the physiological function of this receptor, we have generated a mutant mouse line with a targeted disruption of the GPR54 receptor (GPR54 -/-). The analysis of the GPR54 mutant mice revealed developmental abnormalities of both male and female genitalia and histopathological changes in tissues which normally contain sexually dimorphic features. These data suggest a role for GPR54/KiSS-1 in normal sexual development, and indicate that study of the GPR54 mutant mice may provide valuable insights into human reproductive syndromes.     

Design and synthesis of fluorescent probes for GPR54

Kisspeptins are neuropeptides that induce the secretion of gonadotropin-releasing hormone via the activation of the cognate receptor, G-protein coupled receptor 54 (GPR54). The kisspeptin–GPR54 axis is associated with the onset of puberty and the maintenance of the reproductive system. In this study, several fluorescent probes have been designed and synthesized for rat GPR54 through the modification of the N-terminus of rat kisspeptins to allow for the visualization of the expression and localization of kisspeptin receptor(s) in living cells and native tissues. The tetramethylrhodamine (TMR) and rhodamine green (RG)-labeled kisspeptins exhibited good binding and agonistic activities towards GPR54, and the results of the application studies demonstrated that these fluorescent probes could be used effectively for the detection of GPR54 receptors in flow cytometry and confocal microscopy experiments.     

Differential ovarian expression of KiSS-1 and GPR-54 during the estrous cycle and photoperiod induced recrudescence in Siberian hamsters (Phodopus sungorus)

Kisspeptins, coded by the KiSS-1 gene, regulate aspects of the reproductive axis by stimulating GnRH release via the G protein coupled receptor, GPR54. Recent reports show that KiSS/GPR54 may be key mediators in photoperiod-controlled reproduction in seasonal breeders, and that KiSS-1/GPR54 are expressed in the hypothalamus, ovaries, placenta, and pancreas. This study examined the expression of KiSS-1/GPR54 mRNA and protein in ovaries of Siberian hamsters (Phodopus sungorus). Ovaries from cycling hamsters were collected during proestrus (P), estrus (E), diestrus I (DI), and diestrus II (DII). To examine KiSS-1/GPR54 during stimulated recrudescence, additional hamsters were maintained either in long day (LD 16L:8D, control) or short day (SD 8L:16D) for 14 weeks and then transferred to LD for 0-8 weeks. Staining of KiSS-1/GPR54 protein was detected by immunohistochemistry in steroidogenic cells of pre-antral and antral follicles, and corpora lutea. Immunostaining peaked in P and E, but decreased in the diestrus stages (P < 0.05). In recrudescing ovaries, KiSS-1/GPR54 immunostaining was low after 14 weeks of SD exposure (post-transfer [PT] week 0), and increased during the early weeks of recrudescence. Expression of KiSS-1/GPR54 mRNA was low with short day exposure, but increased during recrudescence and was higher at PT week 8 as compared to PT weeks 0 and 2 (P < 0.05). The elevated KiSS-1/GPR54 expression during P and E suggests a potential role in ovulation in Siberian hamsters. Transient increases in KiSS-1/GPR54 expression following LD stimulation are also suggestive of possible involvement in ovulation and/or restoration of ovarian function.     

GPR54 deficiency reduces the Treg population and aggravates experimental autoimmune encephalomyelitis in mice

GPR54 is highly expressed in the central nervous system and plays a crucial role in pubertal development. However, GRP54 is also expressed in the immune system, implying possible immunoregulatory functions. Here we investigated the role of GPR54 in T cell and immune tolerance. GPR54 deficiency led to an enlarged thymus, an increased number of thymocytes, and altered thymic micro-architecture starting around puberty, indicating GPR54 function in T-cell development through its regulatory effect on the gonadal system. However, flow cytometry revealed a significant reduction in the peripheral regulatory T cell population and a moderate decrease in CD4 single-positive thymocytes in prepubertal Gpr54~(-/-) mice. These phenotypes were confirmed in chimeric mice with GPR54 deficient bone marrow-derived cells. In addition, we found elevated T cell activation in peripheral and thymic T cells in Gpr54~(-/-) mice. When intact mice were immunized with myelin oligodendrocyte glycoprotein, a more severe experimental autoimmune encephalomyelitis(EAE) developed in the Gpr54~(-/-) mice. Interestingly, aggravated EAE disease was also manifested in castrated and bone marrow chimeric Gpr54~(-/-) mice compared to the respective wild-type control,suggesting a defect in self-tolerance resulting from GPR54 deletion through a mechanism that bypassed sex hormones. These findings demonstrate a novel role for GPR54 in regulating self-tolerant immunity in a sex hormone independent manner.     

Kiss1和GPR54基因多态性与多囊卵巢综合征相关性分析

本研究旨在探讨Kiss1和GPR54基因多态性与多囊卵巢综合征的相关性。利用超声检查卵巢体积、血清睾酮、游离雄激素指数情况;临床评估患者身高(cm)和体重(kg)、BMI、静息血压、痤疮和黑棘皮病的分布;ELISA酶联免疫法检测血清中的kisspeptin和睾酮水平,使用Next generation sequencing方法(LGC group, Germany)对基因(Kiss1, GPR54)进行测序。结果显示,PCOS患者比对照组女性具有更高的BMI和mFG评分,PCOS患者血清Kisspeptin和睾酮浓度显著提高,且LH浓度也显著高于对照组(p<0.05)。GPR54和Kiss1 2个基因在患者体内存在多态性;测序分析结果显示GPR54基因存在的2个新的SNP位点(chr19:918686, A→G和chr19:918735, A→G),这2个新的多态性位于内含子区域(内含子2),Kiss1基因也存在两个SNP,位于非翻译变体5的末端(rs5780218)和外显子3 (rs4889),即GPR54基因存在A→G多态性,Kiss1基因为CTT→CT/G→C多态性,且相关性关联分析结果表明,GPR54基因型多态性(Chr19:918735)与PCOS风险增加相关(p<0.05);而Kiss1 SNP的基因型与PCOS风险之间没有关联。此外,PCOS与GPR54和Kiss1基因的单倍型没有显著关联。本研究推论对PCOS发生风险的遗传影响可能不仅是通过直接改变Kiss1/GPR54相互作用,而且还可能通过改变个体与环境因素的相互作用。     

Flt3 mutation activates p21WAF1/CIP1 gene expression through the action of STAT5

Metastin, a post-translationally modified variant of KiSS1, was recently identified as an endogenous peptide agonist for a novel G-protein coupled receptor, hOT7T175 (AXOR12, GPR54). In this study, we analyzed the role of KiSS1 and hOT7T175 in both pancreatic cancer tissues and pancreatic cancer cell lines. Furthermore, we synthesized novel short variant forms of metastin and tested the inhibitory effect of those variants on in vitro cell functions that are relevant to metastasis. Pancreatic cancer tissues showed significantly lower expression of KiSS1 mRNA than normal tissues (p=0.018), while cancer tissues showed significantly higher expression of hOT7T175 mRNA than normal pancreatic tissues (p=0.027). In human pancreatic cancer cell lines, KiSS1 mRNA was highly expressed in 2 out of 6 pancreatic cancer cell lines, while hOT7T175 mRNA was expressed in all cell lines at various degrees. PANC-1 cells showed the highest expression of hOT7T175. Exogenous metastin did not suppress cell proliferation but significantly reduced the in vitro migration of PANC-1 cells (p<0.01). Metastin induced activation of ERK1 in PANC-1 and AsPC-1 cells. Finally, we synthesized 3 novel short variant forms of metastin, FM053a2TFA, FM059a2TFA, and FM052a4TFA. These metastin variants significantly suppressed the migration of PANC-1 cells and activated ERK1. These data suggest that the metastin receptor, hOT7T175, is one of the promising targets for suppression of metastasis, and that small metastin variants could be an anti-metastatic agent to pancreatic cancer.     

KISS-1/GPR54基因及其在生殖中的作用

KISS-1及其受体GPR54基因对青春期的正常启动具有重要作用。青春期开始前后, 动物下丘脑中KISS-1和GPR54 mRNA水平很高, Kisspeptins(KISS-1基因产物)通过激活GPR54增加促性腺激素的释放, KISS-1基因的表达受性腺类固醇激素的调控。GPR54基因突变可以导致人和鼠的特发性促性腺激素分泌不足性腺机能减退症和促性腺激素依赖性性早熟。文章还介绍了KISS-1、GPR54基因的结构、表达、多态性以及和其它生殖调控因子之间的相互关系。     

The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54.

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.     

Postmenopausal increase in KiSS-1, GPR54, and luteinizing hormone releasing hormone (LHRH-1) mRNA in the basal hypothalamus of female rhesus monkeys

The G-protein coupled receptor, GPR54, and its ligand, kisspeptin-54 (a KiSS-1 derived peptide) have been reported to be important players in control of LHRH-1 release. However, the role of the GPR54 signaling in primate reproductive senescence is still unclear. In the present study we investigated whether KiSS-1, GPR54, and LHRH-1 mRNA in the brain change after menopause in female rhesus monkeys using quantitative real-time PCR. Results indicate that KiSS-1, GPR54, and LHRH-1 mRNA levels in the medial basal hypothalamus (MBH) in postmenopausal females (28.3+/-1.1 years of age, n=5) were all significantly higher than that in eugonadal adult females (14.7+/-2.1 years of age, n=9), whereas KiSS-1, GPR54, and LHRH-1 mRNA levels in the preoptic area (POA) did not have any significant changes between the two age groups. To further determine the potential contribution by the absence of ovarian steroids, we compared the changes in KiSS-1, GPR54, and LHRH-1 mRNA levels in young adult ovarian intact vs. young ovariectomized females. Results indicate that KiSS-1 and LHRH-1 mRNA levels in the MBH, not POA, in ovariectomized females were significantly higher than those in ovarian intact females, whereas GPR54 mRNA levels in ovariectomized females had a tendency to be elevated in the MBH, although the values were not quite statistically significant. Collectively, in the primate the reduction in the negative feedback control by ovarian steroids appears to be responsible for the aging changes in kisspeptin-GPR54 signaling and the elevated state of the LHRH-1 neuronal system.     

Expression of a functional g protein-coupled receptor 54-kisspeptin autoregulatory system in hypothalamic gonadotropin-releasing hormone neurons

The G protein-coupled receptor 54 (GPR54) and its endogenous ligand, kisspeptin, are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons with primers for GnRH, kisspeptin-1, and GPR54 revealed expression of all three gene products. Also, constitutive and GnRH agonist-induced bioluminescence resonance energy transfer between Renilla luciferase-tagged GnRH receptor and GPR54 tagged with green fluorescent protein, expressed in human embryonic kidney 293 cells, revealed heterooligomerization of the two receptors. Whole cell patch-clamp recordings from identified GnRH neurons showed initial depolarizing effects of kisspeptin on membrane potential, followed by increased action potential firing. In perifusion studies, treatment of GT1-7 neuronal cells with kisspeptin-10 increased GnRH peak amplitude and duration. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1-7 cells were detected by a specific RIA and was significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. Stimulation of GnRH secretion by kisspeptin and the opposing effects of GnRH on kisspeptin secretion indicate that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to regulate GnRH and kisspeptin secretion from GnRH neurons.