Activation of G-protein-coupled receptor 39 reduces neuropathic pain in a rat model

Activated G-protein-coupled receptor 39(GPR39)has been shown to attenuate inflammation by interacting with sirtuin 1(SIRT1)and peroxisome proliferator-activated receptor-γcoactivator 1α(PGC-1α).However,whether GPR39 attenuates neuropathic pain remains unclear.In this study,we established a Sprague-Dawley rat model of spared nerve injury-induced neuropathic pain and found that GPR39 expression was significantly decreased in neurons and microglia in the spinal dorsal horn compared with sham-operated rats.Intrathecal injection of TC-G 1008,a specific agonist of GPR39,significantly alleviated mechanical allodynia in the rats with spared nerve injury,improved spinal cord mitochondrial biogenesis,and alleviated neuroinflammation.These changes were abolished by GPR39 small interfering RNA(siRNA),Ex-527(SIRT1 inhibitor),and PGC-1αsiRNA.Taken together,these findings show that GPR39 activation ameliorates mechanical allodynia by activating the SIRT1/PGC-1αpathway in rats with spared nerve injury.

MicroRNA-760 acts as a tumor suppressor in gastric cancer development via inhibiting G-protein-coupled receptor kinase interacting protein-1 transcription

BACKGROUND Gastric cancer(GC)has become a serious threat to people's health.Accumulative evidence reveals that dysregulation of numerous microRNAs(miRNAs)has been found during malignant formation.So far,the role of microRNA-760(miR-760)in the development of GC is largely unknown.AIM To measure the expression level of miR-760 in GC and investigate its role in gastric tumorigenesis.METHODS Real-time quantitative polymerase chain reaction and Western blot analysis were used to measure the expression of miR-760 and G-protein-coupled receptor kinase interacting protein-1(GIT1).Cell growth was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)and cell colony formation assays.Apoptosis was assessed by flow cytometric analysis.The relationship between miR-760 and GIT1 was verified by luciferase reporter assay.RESULTS The results showed that the expression of miR-760 was decreased in GC and associated with poor clinical outcomes in GC patients.Furthermore,miR-760 restrained cell proliferation and cell colony formation and induced apoptosis in GC cells.In addition,miR-760 directly targeted GIT1 and negatively regulated its expression in GC.GIT1 was upregulated in GC and predicted a worse prognosis in GC patients.We also found that upregulation of GIT1 weakened the inhibitory CONCLUSION In conclusion,miR-760 targets GIT1 to inhibit cell growth and promote apoptosis in GC cells.Our data demonstrate that miR-760 may be a potential target for the treatment of GC.

Desensitization of G-protein-coupled receptors induces vascular hypocontractility in response to norepinephrine in the mesenteric arteries of cirrhotic patients and rats

BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.

Role of doublecortin-like kinase 1 and leucine-rich repeat-containing G-protein-coupled receptor 5 in patients with stage Ⅱ/Ⅲ colorectal cancer:Cancer progression and prognosis

BACKGROUND Cancer stem cells(CSCs)are a subpopulation of cancer cells with the potential of self-renewal and differentiation.CSCs play critical roles in tumorigenesis,recurrence,metastasis,radiation tolerance and chemoresistance.AIM To assess the expression patterns and clinical potential of doublecortin-like kinase 1(DCLK1)and leucine-rich repeat-containing G-protein-coupled receptor 5(Lgr5),as prognostic CSC markers of colorectal cancer(CRC).METHODS The expression of DCLK1 and Lgr5 in CRC tissue sections from 92 patients was determined by immunohistochemistry.Each case was evaluated using a combined scoring method based on signal intensity staining(scored 0-3)and the proportion of positively stained cancer cells(scored 0-3).The final staining score was calculated as the intensity score multiplied by the proportion score.Low expression of DCLK1 and Lgr5 was defined as a score of 0-3;high expression of DCLK1 and Lgr5 was defined as a score of≥4.Specimens were categorized as either high or low expression,and the correlation between the expression of DCLK1 or Lgr5 and clinicopathological factors was investigated.RESULTS DCLK1 and Lgr5 expression levels were significantly positively correlated.CRC patients with high DCLK1,Lgr5 and DCLK1/Lgr5 expressions had poorer progression-free survival and overall survival.Moreover,high expression of DCLK1 was an independent prognostic factor for recurrence and overall survival in patients with CRC by multivariate analysis(P=0.026 and P=0.049,respectively).CONCLUSION DCLK1 may be a potential CSC marker for the recurrence and survival of CRC patients.

利用深度迁移学习靶向GPCRs的配体活性预测

G蛋白偶联受体(GPCRs)是最重要的药物靶标之一,约占市场上药物靶标的34%。药物发现过程中,配体生物活性的准确建模和解释对于筛选苗头化合物至关重要。研究表明,同源的G蛋白偶联受体能提升配体分子生物活性的预测性能和可解释性。提出了一种新的方法GLEM,用多任务下的深度迁移学习来预测配体的生物活性,并通过组稀疏来识别相关的关键子结构。GLEM方法在9组30个具有代表性的人类GPCR数据集上进行了实验,这些GPCRs涵盖了大部分人类GPCRs的子家族,每个GPCR数据集都包含60~3000个配体。实验结果表明,GLEM方法在绝大多数数据集中都获得了最好的性能。与单任务学习方法相比,GLEM方法在r2上平均提升了31.72%;与深度学习方法相比,GLEM方法在r2上平均提升了22.45%。此外,还评估了不同数量的训练样本对模型性能的影响,实验发现GLEM方法在小样本情况下表现最好。

Peripheral actions and direct central-local communications of melanocortin 4 receptor signaling

Melanocortin 4 receptor(MC4R),the most important monogenetic cause of human metabolic disorders,has been of great interest to many researchers in the field of energy homeostasis and public health.Because MC4R is a vital pharmaceutical target for maintaining controllable appetite and body weight for professional athletes,previous studies have mainly focused on the central,rather than the peripheral,roles of MC4R.Thus,the local expression of MC4R and its behavioral regulation remain unclear.In an attempt to shed light on different directions for future studies of MC4R signaling,we review a series of recent and important studies exploring the peripheral functions of MC4R and the direct physiological interaction between peripheral organs and central MC4R neurons in this article.

β-Arrestins参与GPCRs信号通路的分子机制

β-抑制蛋白(β-arrestins)是一类在β肾上腺素受体激酶(βARK)提纯过程中发现的重要支架蛋白和信号调控因子;G蛋白偶联受体(GPCRs)为7次跨膜受体,在细胞信号转导中发挥关键作用,是很多临床药物的作用靶点.β-抑制蛋白作为衔接蛋白,调控GPCRs相关的信号通路,介导GPCRs的脱敏、内化、循环、复敏等生理过程,影响多种疾病的进程.本文总结了β-抑制蛋白参与GPCRs信号通路的研究进展,侧重阐明了其中的分子机制,以期为开发新一代调控GPCRs功能活性的相关药物提供理论基础.

基于多视图矩阵补全的蛋白受体功能预测

蛋白受体是细胞信号转导的重要组成部分,也是人类最重要的药物靶点,其中G蛋白偶联受体(G Protein Coupled Receptors,GPCRs)占绝大部分,目前市场上大约34%的药物都以GPCRs作为靶点.准确地注释GPCR蛋白的生物学功能对于理解它们涉及的生理过程及靶向药物发现至关重要,其中基因本体学(Gene Ontology,GO)是描述蛋白质功能最常用的方式,GPCR蛋白和GO都包含多个视图信息,有效利用这些信息可有效提升蛋白质功能的预测性能.因此,提出一种基于多视图的归纳矩阵补全方法MVIMC(Multi-View Inductive Matrix Completion)来预测GPCR蛋白的GO生物学功能.MVIMC有效利用了GPCR蛋白和GO标记视图信息,其中GPCR包含文本信息和结构域信息,GO包含文本信息.实验结果表明,MVIMC在分子功能和生物过程两方面的预测概率分别达到68%和69%,优于目前最好的矩阵补全方法以及CAFA蛋白质功能预测比赛中的常用方法.

稻瘟病菌cAMP受体类GPCR的生物信息学分析

G蛋白偶联受体(GPCRs)是一类重要的细胞表面受体,通过G蛋白介导着多种生物学途径,其介导的信号转导机制及其作为药物靶标的研究已成为研究热点之一。运用生物信息学的方法,对稻瘟病菌全基因组序列中可能的cAMP类GPCR基因进行了生物信息学分析,为深入研究cAMP类GPCR基因的结构和生物学功能奠定了基础。

醛固酮瘤(APA)发病相关的G蛋白耦联受体(GPCRs)研究进展

醛固酮瘤（aldosterone-producing adenoma,APA）是原发性醛固酮增多症的一个重要亚型,约占30％～60％,是引起继发性高血压的重要病因.有关APA的发病机制,可见不同水平与角度的研究,但是对于APA的具体发病机制仍不清楚.本文就已知的与发病相关的G蛋白耦联受体（G-protein-coupled receptors,GPCRs）进行论述.

Conformational dynamics in GPCR signaling by NMR

G-protein-coupled receptors(GPCRs)mediate a wide range of cellular responses to various ligands or stimuli,and are the most important drug targets associated with human diseases.While major advances in GPCR structural biology have greatly deepened our understanding of its activation mechanism,the highly complex changes in the structural dynamics of GPCRs during activation remain underdetermined and their links to physiological functions largely unknown.Solution nuclear magnetic resonance(NMR)spectroscopy is an essential technique that allows the characterization of protein structural dynamics at atomic level,and has been applied in the studies of GPCR structural-function relationship in the past decade.Herein,we summarize a few specific studies in which solution NMR methods were employed and provided novel insights into questions difficult to be addressed by other methods.

Activation of β_2-Adrenergic Receptor Induced by Three Catecholamine Agonists:a Docking and Molecular Dynamics Study

We studied the activation of β2-adrenergic receptor(β2 AR) by norepinephrine,epinephrine and isoproterenol using docking and molecular dynamics(MD) simulation.The simulation was done on the assumption that β2 AR was surrounded with explicit water and infinite lipid bilayer membrane at body temperature.So the result should be close to that under the physiological conditions.We calculated the structure of binding sites in β2 AR for the three activators.We also simulated the change of the conformation of β2 AR in the transmembrane regions(TMs),in the molecular switches,and in the conserved DRY(Aspartic acid,Arginine and Tyrosine) motif.This study provides detailed information concerning the structure of β2 AR during activation process.

共表达人μ阿片受体与Gq蛋白的稳定细胞株的建立及功能鉴定

建立共表达人μ阿片受体(humanμ-opiatereceptor,hMOR,OPRM)与Gq蛋白的CHO-Flp In稳定细胞模型,并鉴定其药理学功能,可为体外高通量筛选靶向OPRM的药物奠定基础。该研究首先构建重组表达质粒OPRM-pcDNA5/FRT,并进行鉴定;然后通过脂质体转染法将OPRM-pcDNA5/FRT、GqG66Di5-pIRES/puro3和FLP重组酶质粒POG44共转染CHO-Flp In细胞,经抗性加压和有限稀释法挑取耐药单克隆,采用FLIPR钙信号检测方法筛选阳性克隆株;最后,通过RT-q PCR对细胞中的OPRM和GqG66Di5m RNA表达水平进行检测,并选用OPRM激动剂DAMGO和抑制剂Naloxone对稳定细胞株的药理学功能进行鉴定。结果显示:经酶切确定了重组质粒的正确构建;通过重组质粒转染、抗生素加压筛选以及钙信号测定获得22个具有活性的克隆细胞株,其中15号细胞株的荧光信号值最高,命名为Gq-OPRM1-CHO;与对照组相比,Gq-OPRM1-CHO细胞组中OPRM与GqG66Di5基因m RNA水平分别升高约400倍和120倍;在Gq-OPRM1-CHO细胞中,FLIPR钙信号检测激动剂DAMGO的EC_(50)为0.02±0.002μmol/L,抑制剂Naloxone的IC_(50)为0.04±0.003μmol/L。该研究成功建立了OPRM与GqG66Di5蛋白稳定共表达的细胞模型Gq-OPRM1-CHO,该细胞株具有对OPRM激动剂和拮抗剂特异性反应的药理学功能。

味觉受体及其传感器研究与应用

中国传统饮食和食品的烹制与加工讲究色、香、味俱全,其中"酸、甜、苦、辣、咸五味调和"又构成其核心标准。国际上也分为"五味",分别为"酸、甜、苦、咸、鲜",其中少了辣味,多了鲜味。也有人建议将脂肪的味道定义为"香"味,但是中国人的"香"实际上是"香气",属于嗅觉,而非味觉。大量研究证明,多数味觉受体作为营养传感系统,如苦、甜、鲜都属于G蛋白偶联受体超家族成员,而且其分布并不限于味蕾、肠道等,其他组织均有分布,是药物筛选的重要靶标。然而,到目前为止,市场上进行味觉测定仍然依赖于电子鼻和电子舌等仪器设备,味觉受体传感器及其有关技术则仍处于探索和研究阶段。其主要原因是:味觉受体和其他大多数受体一样,在与配体识别和启动信号传递时主要依赖于弱相互作用,如何将这些弱相互作用转变为传感器可以处理并放大的光、声、电、磁、热等信号,从而实现其定量测定是一个关键性难题。但是,由于味觉受体在医药筛选、食品添加剂、食品的功能性评价和代谢综合征预防等方面的巨大应用与开发前景,其检测方法一直是科学家关注的焦点之一。本文将针对味觉受体及其传感器检测技术的研究进展进行综合评述,讨论其未来发展和应用前景。

味觉受体及其对食品功能评价的应用潜力

哺乳动物味觉系统传统分类为五味:鲜、甜、咸、酸、苦。这些味觉让我们能够大概了解食物的组成成分。味觉的传感受体经鉴定属于最大的基因家族——G蛋白偶联受体的主要成员T1R和T2R。有研究表明,还有一种和脂肪传感有关的受体,可以介导游离脂肪酸(free fatty acid,FFA)的味道,也属于G蛋白偶联受体(G protein-coupled receptors,GPCR)家族成员。越来越多的研究显示,味觉受体不仅在味觉器官中表达,也广泛表达于其他组织和器官中。但是大多数研究结果都是通过反转录聚合酶链式反应(reverse transcriptionpolymerase chain reaction,RT-PCR)和微阵列方法得到,尚未对其蛋白表达和生理功能进行系统研究。近年来,一些文章报道了关于胃肠道味觉受体的生理功能。这些研究结果进一步证明嗅觉和味觉受体绝不仅仅局限于味觉和嗅觉传感,可能还有其他潜在的重要功能。本文首先整理和总结了嗅觉和味觉受体在嗅觉和味觉器官以外的组织中的表达情况,而后深入探讨其潜在的多种生理功能,从营养传感、吸收控制及对代谢平衡、能量吸收与控制的影响,到代谢紊乱所引起的疾病。最后,预期这些受体及其检测技术的发展方向和应用前景,特别是在食品性味评价和主要功能性成分,如:白藜芦醇、多酚类、黄酮类和其他植物化合物的功能性评价中的应用。

G蛋白偶联受体相关分选蛋白功能特征与相关疾病研究进展

G蛋白偶联受体(G protein-coupled receptors,GPCRs)作为最大的一类膜蛋白受体家族,可被多种配体激活并发挥相应的信号转导功能,参与生物体内重要的生理过程。G蛋白偶联受体相关分选蛋白(G protein-coupled receptors associated sorting proteins,GASPs)则对内吞后的GPCRs分选过程发挥着重要的作用,并介导受体进入降解或再循环途径,进而调控细胞的信号转导等过程。研究发现GASPs的功能缺陷与多种疾病相关,包括神经系统疾病、肿瘤和耳聋等。本文重点介绍了G蛋白偶联受体相关分选蛋白的功能特征及其相关信号通路,描述了GASPs功能缺陷与疾病的关联性及家族蛋白与GPCRs的相互作用、GASPs分选途径的发现、参与的信号通路及对基因转录调控,以期为GASPs相关多种疾病的治疗提供新的思路和策略。

靶向G蛋白偶联受体的高通量药物筛选方法

G蛋白偶联受体(G-protein-coupled receptors,GPCR)是一类具有7次跨膜结构的膜蛋白。GPCR介导多种重要的生理功能,与很多疾病密切相关,是最重要的现代药物靶点家族。目前市场上有近50%的药物是以GPCR为靶点的。因此,GPCR分析方法和GPCR配体筛选方法的研究是当今世界新药研究的重点和热点。本文归纳介绍了近年来被广泛使用的GPCR药物发现方法,以及靶向GPCR高通量筛选技术的最新研究进展。

GPCR-Gs mediates the protective effects of ginsenoside Rb1 against oxygen-glucose deprivation/re-oxygenation-induced astrocyte injury

Objectives:To investigate whether the protective actions of ginsenoside Rb1(Rb1)on astrocytes are mediated through the G_(s)-type G-protein-coupled receptor(GPCR-G_(s)).Methods:Primary astrocyte cultures derived from neonatal mouse brain were used.Astrocyte injury was induced via oxygen-glucose deprivation/re-oxygenation(OGD/R).Cell morphology,viability,lactate dehydrogenase(LDH)leakage,apoptosis,glutamate uptake,and brain-derived neurotrophic factor(BDNF)secretion were assessed to gauge cell survival and functionality.Western blot was used to investigate the cyclic adenosine monophosphate(cAMP)and protein kinase B(Akt)signaling pathways.GPCR-G_(s)-specific inhibitors and molecular docking were used to identify target receptors.Results:Rb1 at concentrations ranging from 0.8 to 5μM did not significantly affect the viability,glutamate uptake,or BDNF secretion in normal astrocytes.OGD/R reduced astrocyte viability,increasing their LDH leakage and apoptosis rate.It also decreased glutamate uptake and BDNF secretion by these cells.Rb1 had protective effects of astrocytes challenged by OGD/R,by improving viability,reducing apoptosis,and enhancing glutamate uptake and BDNF secretion.Additionally,Rb1 activated the cAMP and Akt pathways in these cells.When the GPCR-G_(s) inhibitor NF449 was introduced,the protective effects of Rb1 completely disappeared,and its activation of cAMP and Akt signaling pathways was significantly inhibited.Conclusion:Rb1 protects against astrocytes from OGD/R-induced injury through GPCR-G_(s) mediation.

果蝇气味受体复合物Or22a-Or83b研究进展

昆虫的气味受体长久以来被认为是一种G蛋白偶联受体,遵循从线虫到人类的通用模式。但最近的研究显示,果蝇的气味探测与一种蛋白复合体有关,该复合体由一个调节型气味受体,例如Or22a,加上离子通道Or83b,构成一个反转的G蛋白偶联受体构象。调节型气味Or22a受体结合气体分子产生第二信使,并促使Or83b进一步作用。基于此结构基础,揭示出了昆虫气味探测与相关反应的新模式和机理。