Identification of a G protein-coupled receptor for 11(R),12(S)-epoxyeicosatrienoic acid and its effects in the cardiovascular system

11(R),12(S)-环氧二十碳三烯酸G蛋白偶联受体的鉴定及其对心血管系统的影响

• 批准号: 316581735
• 负责人: Professorin Dr. Ingrid Fleming, Ph.D.
• 金额: --
• 依托单位: Vascular Research Center
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/316581735?language=en
• 关键词: Identification; protein; coupled; receptor; 11

Epoxyeicosatrienoic acids (EETs) are generated from arachidonic acid by cytochrome P450 (CYP) enzymes as different regioisomers and play a central role in cellular processes including Ca2+ signalling, hyperpolarization, intracellular communication, angiogenesis, and the prevention or resolution of inflammation as well as the control of vascular tone and blood pressure. Work from our group has demonstrated that 11,12-EET seems to be the biologically more active of the regioisomers and able to elicit rapid cellular responses, the fastest in endothelial cells being the translocation of transient receptor (TRP) C3/C6 channels to caveolae. Exactly how 11,12-EET initiates cell signalling has yet to be resolved. Despite this there is a considerable amount of circumstantial evidence to indicate that a specific G protein coupled membrane receptor for 11,12-EET; more specifically 11(R),12(S)-EET, exists: (1) high affinity EET binding sites have been detected on the surface of some cells, (2) different EET stereoisomers, and regioisomers show distinct biological effects, (3) stable and specific EET agonists and antagonists have been generated to mimic or prevent the actions of 11,12-EET, (4) 11,12-EET induced cellular responses; such as cell proliferation, gap junctional communication or TRP channel translocation, are absolutely reliant on the activation of protein kinase A (PKA), moreover, (5) the downregulation of Gs but not Gq/11 proteins abolishes endothelial cell sensitivity to 11,12-EET. Identifying a receptor for 11(R),12(S)-EET receptor is not straight forward given that the agonist is a fatty acid epoxide, this means that many classical binding studies and labelling approaches used to identify peptide receptors are not easily applicable. Also although many of the effects of the EETs rely on PKA activation, the EET-induced changes in cyclic AMP tend to be small and inconsistent, particularly in endothelial cells.In preliminary studies it has been possible to demonstrate GPR124 as a candidate 11,12-EET receptor in endothelial cells. Indeed, the downregulation of GPR124 abolishes the 11,12-EET-induced translocation of TRPC6 channels as well as 11,12-EET-induced endothelial cell proliferation (responses to VEGF were unaffected) and endothelial cell sprouting. These effects correlated with changes in the cellular distribution of the receptor. Our hypothesis is that GPR124 is the receptor for 11(R),12(S)-EET and is required for the biological actions of the epoxide in endothelial cells. The aims of this project are to (1) determine whether GPR124 displays characteristics of a receptor for 11,12-EET by assessing its sensitivity to EET analogues (so called EET agonists and antagonists), as well as receptor desensitization, internalization and recycling, (2) determine the downstream signals activated by GPR124 in the presence of 11,12-EET, and (3) to determine whether the deletion of GPR124 in adult mice alters biological responses to 11,12-EET.

环氧二十碳三烯酸(EETs)是由细胞色素P450(CYP)酶以不同区域异构体的形式由花生四烯酸合成的，在细胞内钙信号、超极化、细胞内通讯、血管生成、预防或消除炎症以及控制血管张力和血压等过程中发挥着核心作用。我们小组的工作表明，11，12-EET似乎是区域异构体中生物学上更活跃的，能够引起快速的细胞反应，在内皮细胞中，最快的是瞬时受体(Trp)C3/C6通道移位到小窝。11，12-EET是如何启动细胞信号转导的还有待解决。尽管如此，有大量的间接证据表明，11，12-EET的G蛋白偶联膜受体，更具体地说，11(R)，12(S)-EET，存在：(1)在一些细胞表面检测到高亲和力的EET结合部位，(2)不同的EET立体异构体，并且区域异构体显示出不同的生物学效应，(3)已经产生了稳定和特异的EET激动剂和拮抗剂来模拟或阻止11，12-EET，(4)11，12-EET诱导的细胞反应；如细胞增殖、缝隙连接通讯或Trp通道转位，都完全依赖于蛋白激酶A(PKA)的激活。此外，(5)Gs蛋白的下调而不是GQ/11蛋白的下调使内皮细胞对11，12-EET的敏感性消失。确定11(R)，12(S)-EET受体的受体并非易事，因为激动剂是脂肪酸环氧化物，这意味着许多用于识别多肽受体的经典结合研究和标记方法并不容易应用。此外，尽管EET的许多作用依赖于PKA的激活，但EET诱导的环状AMP的变化往往很小且不一致，特别是在内皮细胞中。在初步研究中，GPR124可能是内皮细胞中11，12-EET的候选受体。事实上，GPR124的下调取消了11，12-EET诱导的TRPC6通道的移位以及11，12-EET诱导的内皮细胞增殖(对血管内皮生长因子的反应不受影响)和内皮细胞的萌发。这些效应与受体细胞分布的变化有关。我们的假设是GPR124是11(R)，12(S)-EET的受体，是环氧化物在内皮细胞中发挥生物学作用所必需的。本项目的目的是(1)通过评估GPR124对EET类似物(所谓的EET激动剂和拮抗剂)的敏感性以及受体的脱敏、内化和再循环来确定GPR124是否显示出11，12-EET受体的特征，(2)确定GPR124在11，12-EET存在的情况下激活的下游信号，以及(3)确定成年小鼠GPR124的缺失是否改变了对11，12-EET的生物反应。