Increased endothelin receptor B and G protein coupled kinase-2 in the mesentery of portal hypertensive rats

AIM: To elucidate the mechanisms of mesenteric vasodilation in portal hypertension (PHT), with a focus on endothelin signaling. METHODS: PHT was induced in rats by common bile duct ligation (CBDL). Portal pressure (PP) was measured directly via catheters placed in the portal vein tract. The level of endothelin-1 (ET-1) in the mesenteric circulation was determined by radioimmunoassay, and the expression of the endothelin A receptor (ETAR) and endothelin B receptor (ETBR) was assessed by immunofluorescence and Western blot. Additionally, expression of G protein coupled kinase-2 (GRK2) and β-arrestin 2, which influence endothelin receptor sensitivity, were also studied by Western blot. RESULTS: PP of CBDL rats increased significantly (11.89 ± 1.38 mmHg vs 16.34 ± 1.63 mmHg). ET-1 expression decreased in the mesenteric circulation 2 and 4 wk after CBDL. ET-1 levels in the systemic circulation of CBDL rats were increased at 2 wk and decreased at 4 wk. There was no change in ETAR expression in response to CBDL; however, increased expression of ETBR in the endothelial cells of mesenteric arterioles and capillaries was observed. In sham-operated rats, ETBR was mainly expressed in the CD31+ endothelial cells of the arterioles. With development of PHT, in addition to the endothelial cells, ETBR expression was noticeably detectable in the SMA+ smooth muscle cells of arterioles and in the CD31+ capillaries. Following CBDL, increased expression of GRK2 was also found in mesenteric tissue, though there was no change in the level of β-arrestin 2. CONCLUSION: Decreased levels of ET-1 and increased ETBR expression in the mesenteric circulation following CBDL in rats may underlie mesenteric vasodilation in individuals with PHT. Mechanistically, increased GRK2 expression may lead to desensitization of ETAR, as well as other vasoconstrictors, promoting this vasodilatory effect.

Adrenal G protein-coupled receptor kinase-2 in regulation of sympathetic nervous system activity in heart failure

Heart failure(HF), the number one cause of death in the western world, is caused by the insufficient performance of the heart leading to tissue underperfusion in response to an injury or insult. It comprises complex interactions between important neurohormonal mechanisms that try but ultimately fail to sustain cardiac output. The most prominent such mechanism is the sympathetic(adrenergic) nervous system(SNS), whose activity and outflow are greatly elevated in HF. SNS hyperactivity confers significant toxicity to the failing heart and markedly increases HF morbidity and mortality via excessive activation of adrenergic receptors, which are G protein-coupled receptors. Thus, ligand binding induces their coupling to heterotrimeric G proteins that transduce intracellular signals. G protein signaling is turned-off by the agonist-bound receptor phosphorylation courtesy of G protein-coupled receptor kinases(GRKs), followed by βarrestin binding, which prevents the GRK-phosphorylated receptor from further interaction with the G proteins and simultaneously leads it inside the cell(receptor sequestration). Recent evidence indicates that adrenal GRK2 and βarrestins can regulate adrenal catecholamine secretion, thereby modulating SNS activity in HF. The present review gives an account of all these studies on adrenal GRKs and βarrestins in HF and discusses the exciting new therapeutic possibilities for chronic HF offered by targeting these proteins pharmacologically.

Roles of G protein-coupled receptors in inflammatory bowel disease

Inflammatory bowel disease(IBD)is a complex disease with multiple pathogenic factors.Although the pathogenesis of IBD is still unclear,a current hypothesis suggests that genetic susceptibility,environmental factors,a dysfunctional immune system,the microbiome,and the interactions of these factors substantially contribute to the occurrence and development of IBD.Although existing and emerging drugs have been proven to be effective in treating IBD,none can cure IBD permanently.G protein-coupled receptors(GPCRs)are critical signaling molecules implicated in the immune response,cell proliferation,inflammation regulation and intestinal barrier maintenance.Breakthroughs in the understanding of the structures and functions of GPCRs have provided a driving force for exploring the roles of GPCRs in the pathogenesis of diseases,thereby leading to the development of GPCR-targeted medication.To date,a number of GPCRs have been shown to be associated with IBD,significantly advancing the drug discovery process for IBD.The associations between GPCRs and disease activity,disease severity,and disease phenotypes have also paved new avenues for the precise management of patients with IBD.In this review,we mainly focus on the roles of the most studied proton-sensing GPCRs,cannabinoid receptors,and estrogen-related GPCRs in the pathogenesis of IBD and their potential clinical values in IBD and some other diseases.

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.

G protein-coupled estrogen receptor in colon function, immune regulation and carcinogenesis

Estrogens play important roles in the development and progression of multiple tumor types.Accumulating evidence points to the significance of estrogen action not only in tumors of hormonally regulated tissues such as the breast,endometrium and ovary,but also in the development of colorectal cancer(CRC).The effects of estrogens in physiological and pathophysiological conditions are mediated by the nuclear estrogen receptorsαandβ,as well as the membranebound G protein-coupled estrogen receptor(GPER).The roles of GPER in CRC development and progression,however,remain poorly understood.Studies on the functions of GPER in the colon have shown that this estrogen receptor regulates colonic motility as well as immune responses in CRC-associated diseases,such as Crohn’s disease and ulcerative colitis.GPER is also involved in cell cycle regulation,endoplasmic reticulum stress,proliferation,apoptosis,vascularization,cell migration,and the regulation of fatty acid and estrogen metabolism in CRC cells.Thus,multiple lines of evidence suggest that GPER may play an important role in colorectal carcinogenesis.In this review,we present the current state of knowledge regarding the contribution of GPER to colon function and CRC.

New insights into sodium transport regulation in the distal nephron:Role of G-protein coupled receptors

The renal handling of Na^+ balance is a major determinant of the blood pressure(BP) level. The inability of the kidney to excrete the daily load of Na+ represents the primary cause of chronic hypertension. Among the different segments that constitute the nephron, those present in the distal part(i.e., the cortical thick ascending limb, the distal convoluted tubule, the connecting and collecting tubules) play a central role in the fine-tuning of renal Na^+ excretion and are the target of many different regulatory processes that modulate Na^+ retention more or less efficiently. G-protein coupled receptors(GPCRs) are crucially involved in this regulation and could represent efficient pharmacological targets to control BP levels. In this review, we describe both classical and novel GPCR-dependent regulatory systems that have been shown to modulate renal Na^+ absorption in the distal nephron. In addition to the multiplicity of the GPCR that regulate Na^+ excretion, this review also highlights the complexity of these different pathways, and the connections between them.

Isoleucine, an Essential Amino Acid, Induces the Expression of Human <i>β</i>Defensin 2 through the Activation of the G-Protein Coupled Receptor-ERK Pathway in the Intestinal Epithelia

Anti-microbial peptides are essential for the intestinal innate immunity that protects the intestinal epithelia from attacks by foreign pathogens. Human β-defensin (HBD) is one of the pivotal anti-microbial peptides that are expressed in the colonic epithelia. This study investigated the effect and the signaling mechanism of inducible β-defensin HBD2 by an essential amino acid, isoleucine (Ile) in colonic epithelial cells. Here we examined the expression level of HBD2 on induction of Ile in epithelial cells, and checked this pathway. HBD2 mRNA was induced by co-incubation with IL-1α and Ile in Caco2 cells, but not by Ile alone. An inhibitor of either ERK or Gi, a subunit of G-proteins, reduced the induction of HBD2 mRNA by Ile. The treatment with Ile also increased the intracellular calcium ion concentration, thus suggesting that the GPCR and ERK signaling pathway mediate the effects of Ile. These results indicate that an essential amino acid, Ile, enhances the expression of an inducible β-defensin, namely HBD2, by IL-1α through the activation of GPCRs and ERK signaling pathway. The administration of Ile may therefore represent a possible option to safely treat intestinal inflammation.

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.

Physiological and pharmacological functions of G protein coupled receptor 124:A review

G protein-coupled receptors(GPCRs)are the largest protein superfamily in the body,expressed in various tissues and organs,and are currently one of the most important clinical drug targets.Recently,a class of GPCRs without endogenous ligands(orphan GPCRs)have been discovered.They exhibit different physiological functions in the body and act extensively on the cardiovascular and cerebrovascular systems.Among them,G protein-coupled receptor 124(GPR124)is an orphaned member of the G protein coupled receptor adhesion family that has attracted much attention.It plays a key role in promoting cerebral angiogenesis and maintaining the stability of the blood-brain barrier.It also associated with cardiovascular and cerebrovascular diseases such as cerebral ischemia and atherosclerosis.However,the role of GPR124 in these diseases,the associated signaling pathways,and possible drug intervention targets are still unclear.This article summarizes the physiological effects,pharmacological effects and related signal pathways of GPR124 published in the field of cardiovascular and cerebrovascular diseases published in recent years,in order to provide a reference for the study of the role of GPR124 in the occurrence and development of diseases.

Overexpression of G protein-coupled receptor 31 as a poor prognosticator in human colorectal cancer

AIM To investigate the expression of G protein-coupled receptor 31 (GPR31) and its clinical significance in human colorectal cancer (CRC).METHODS To determine the association between the GPR31 expression and the prognosis of patients, we obtained paraffin-embedded pathological specimens from 466 CRC patients who underwent initial resection. A total of 321 patients from the First Affiliated Hospital of Sun Yat-sen University from January 1996 to December 2008 were included as a training cohort, whereas 145 patients from the Sixth Affiliated Hospital of Sun Yat-sen University from January 2007 to November 2008 were included as a validation cohort. We examined GPR31 expression levels in CRC tissues from two independent cohorts via immunohistochemical staining. All patients were categorized into either a GPR31 low expression group or a GPR31 high expression group. The clinicopathological factors and the prognosis of patients in the GPR31 low expression group and GPR31 high expression group were compared.RESULTS We compared the clinicopathological factors and the prognosis of patients in the GPR31 low expression group and GPR31 high expression group. Significant differences were observed in the number of patients in pM classification between patients in the GPR31 low expression group and GPR31 high expression group (P = 0.007). The five-year survival and tumor-free survival rates of patients were 84.3% and 82.2% in the GPR31 low expression group, respectively, and both rates were 59.7% in the GPR31 high expression group (P < 0.05). Results of the Cox proportional hazard regression model revealed that GPR31 upregulation was associated with shorter overall survival and tumor-free survival of patients with CRC (P < 0.05). Multivariate analysis identified GPR31 expression in colorectal cancer as an independent predictive factor of CRC patient survival (P < 0.05).CONCLUSION High GPR31 expression levels were found to be correlated with pM classification of CRC and to serve as an independent predictive factor of poor survival of CRC patients.

Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes

In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selec- tivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disorders of the central nervous system. Specifically, new possibilities are explored in relation to allosteric and or- thosteric binding sites on dopamine receptors for the treatment of Parkinson＇s disease, and on muscarinic receptors for Alzheimer＇s disease. Future research can seek to identify ligands that can bind to more than one site on the same receptor, or simultaneously bind to two receptors and form a dimer. For example, the design of bivalent drugs that can reach homo/hetero-dimers of D2 dopa- mine receptor holds promise as a relevant therapeutic strategy for Parkinson＇s disease. Regarding the treatment of Alzheimer＇s disease, the design of dualsteric ligands for mono-oligomeric mus- carinic receptors could increase therapeutic effectiveness by generating potent compounds that could activate more than one signaling pathway.

Functionally diverse ligands modulate different activation states of the formyl peptide receptor 2,a G protein-coupled receptor

OBJECTIVE To identify the mechanisms by which the formyl peptide receptor 2(FPR2)mediates both inflammatory and anti-inflammatory signaling in an agonist-dependent manner.METHODS Cells expressing FPR2 were incubated with weak agonists,Aβ42 and Ac2-26,before stimulation with a strong agonist,WKYMVm.Calcium mobilization,c AMP inhibition and MAP kinase activation were measured.Intramolecular FRET were determined using FPR2 constructs with an ECFP attached to the C-terminus and a Fl As H binding motif embedded in the first or third intracellular loop(IL1 or IL3,respectively).RESULTS Aβ42 did not induce significant Ca^(2+) mobilization,but positively modulated WKYMVm-induced Ca^(2+) mobilization and c AMP reduction in a dose-variable manner within a narrow range of ligand concentrations.Treating FPR2-expressing cells with Ac2-26,a peptide with anti-inflammatory activity,negatively modulated WKYMVm-induced Ca^(2+) mobilization and c AMP reduction.Intramolecular FRET assay showed that stimulation of the receptor constructs with Aβ42 brought the C-terminal domain closer to IL1 but away from IL3.An opposite conformational change was induced by Ac2-26.The FPR2 conformation induced by Aβ42 corresponded to enhanced ERK phosphorylation and attenuated p38 MAPK phosphorylation,whereas Ac2-26 induced FPR2 conformational change corresponding to elevated p38 MAPK phosphorylation and reduced ERK phosphorylation.CONCLUSION Aβ42 and Ac2-26 induce different conformational changes in FPR2.These findings provide a structural basis for FPR2 mediation of inflammatory vs anti-inflammatory functions and identify a type of receptor modulation that differs from the classic positive and negative allosteric modulation.

G蛋白偶联受体(GPCR)腺苷酸环化酶(AcyA)途径介导赭曲霉群体感应现象的机制

赭曲霉(Aspergillus ochraceus)是一种代表性的产毒模式真菌,存在密度依赖性转化的高、低群体密度阈值和携带这种密度信息的群体感应(QS)信号分子--氧脂素。本研究首先基于代表高、低群体密度条件的野生型赭曲霉转录组差异,分析群体密度这一环境条件对赭曲霉行为的影响,结果发现,除了氧脂素代表的细胞通信外,还具有种内对资源的竞争现象,体现在碳代谢。进一步,基于前期实验筛选到负责响应密度信息的膜受体GprC和主要的胞内效应器腺苷酸环化酶(AcyA),构建了基因缺陷型赭曲霉(ΔgprC和ΔacyA)。对比野生型与缺陷型赭曲霉的基因表达及行为差异后发现,GprC-AcyA途径缺陷后不仅中断了密度信息的传递,还直接影响赭曲霉的生命周期。本文研究结果表明,GprC和AcyA作为G蛋白网络的重要枢纽,具有广泛的调控作用,涉及生物过程、细胞组分、分子功能中大部分行为。本研究结果对利用生物手段进行真菌毒素污染的高效、绿色防治具有指导意义。

GPCR二聚体结构及功能

G蛋白偶联受体(G-protein coupled receptor,GPCR)是最广泛表达的膜蛋白家族之一,其可接收胞外信号刺激,通过自身构象变化激活胞内G蛋白等一系列信号通路,参与众多生理调节过程,具有重要的功能,因此其也是重要的药物靶点。GPCR二聚化是调控其功能的重要形式之一,靶向GPCR二聚体开发药物是药物研发的一个新方向。越来越多的研究报道了GPCR二聚化及其结构与功能调控的机制,本文综述了GPCR二聚体结构及功能的研究进展,为了解GPCR二聚体的发现、二聚化方式、功能调控机制,及进一步靶向GPCR二聚体药物开发提供了研究基础。

人源孤儿G蛋白偶联受体hGPCRc的分子克隆及其初步鉴定

孤儿G蛋白偶联受体 (orphanGprotein coupledreceptors ,oGPCRs)是最重要的潜在药物靶点 ,对于创新药物研究意义重大 .根据已有文献及相关基因数据库提供的信息 ,利用RT PCR从人结肠组织获得oGPCR某一成员的氨基酸编码序列 ,大小为 10 14bp ,而且与GenBank已登录序列(AB0 835 98)完全一致 ,称之为hGPCRc ;又用相同的引物以健康志愿者血液基因组DNA作为模板进行PCR扩增 ,亦得到同样大小的DNA序列 ,测序显示二者个别碱基不一致 ,但所对应氨基酸序列并无差异 .另外 ,RT PCR对人源部分组织及细胞系的检测结果显示 :hGPCRc在人脑组织表达最高 ,结肠次之 ,其它组织或细胞系如胃、血液、肝、肺、上皮未检测到该基因的表达 .利用相关软件对hGPCRc分别结果显示 :hGPCRc定位于人染色体 13q32 3,与小鼠、大鼠的对应物序列同源性高达85 % ,但与人源其他已知基因的同源性较低 ,对应的氨基酸序列组成了 7个跨膜区段的结构域 .因此 ,hGPCRc符合GPCR的结构特点 ,应为人类oGPCRs的新成员 .

GPCR偏向性配体介导的选择性功能

G蛋白偶联受体(GPCR)是当今药物治疗中最有效靶向作用的受体超家族之一,它在人类的正常生理状态和疾病过程中都发挥着极大的功效。近年研究发现,GPCR脱敏作用的调节器β-arrestin,可作为真正的衔接蛋白将信号转导到多重效应途径。β-arrestin介导的信号对生化和功能方面的影响力都不同于传统G蛋白介导的信号。由此发现辨别出的多种G蛋白-偏向配体或β-arrestin偏向配体,不仅是用来研究GPCR信号生化特征的有效工具,还具有被开发成治疗药物的潜力。因此,该文就偏向性配体的特点、作用机制、药理学作用及研究偏向性配体的技术进行综述。

以游离脂肪酸为结合配体的GPCRs的研究进展

食物营养成分在肠道中可被分解产生游离脂肪酸.游离脂肪酸除了被吸收氧化分解产生能量供机体利用外,还能通过结合脂肪酸受体激活信号通路,参与多种生理功能的调节,如维持能量平衡、代谢稳态、调节脂质形成与分解、影响机体免疫、结识流动消化成分间接监测菌群数量等.被确认的游离脂肪酸受体包括结合长链脂肪酸的G蛋白偶联受体(GPR)120,结合中链脂肪酸的GPR84,结合短链脂肪酸的GPR41及GPR43.对这些脂肪酸受体的研究可进一步了解肠道脂肪酸的消化与吸收、机体能量平衡与脂质代谢,对增强肠道营养、调节机体免疫和开发针对脂质代谢紊乱疾病的药物有着重要意义.

GPCR跨膜螺旋的结构拓扑建模及其预测方法

7个α跨膜螺旋组成的螺旋束是G蛋白偶联受体的最主要拓扑特征,其三维结构的预测精度直接影响完整受体的三维结构预测、配体对接及功能分析的准确性.近期许多研究小组提出了各种方法,同时也遇到了一个共同的问题:采样时难以在7个跨膜螺旋结构的保守性与局部多样性之间获得平衡,其实质是未将两者统一到一个系统模型中.文中针对跨膜螺旋的空间结构特点,建立了兼顾保守性与多样性的结构拓扑模型,并利用该模型形成了4阶段的结构优化方法,试图获得采样广度与深度的平衡.同时,引入基于结构拓扑的能量项与约束,起到了优化评判标准和剪裁采样空间的作用,有效地预测了跨膜螺旋的三维结构.使用文中方法展开了3组验证实验,用8个已解构的目标分别与GPCRDOCK2010的参赛结果、知名结构预测工具Swiss和MODELLER进行了比较.与Swiss的比较中,文中方法有5个目标获得了更优的三维螺旋结构;与单模板、多模板的MODELLER的比较中,文中方法分别在6个目标与7个目标上取得了优势.

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.