DEVELOPMENT OF A FLUORESCENCE BASED TESTSYSTEM FOR GPCR ACTIVATION

开发基于荧光的 GPCR 激活测试系统

• 批准号: 7601035
• 负责人: MARTIN J LOHSE
• 金额: $ 1.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601035
• 关键词: Adenosine; Adenylate Cyclase; Affect; Behavior; Binding; Catechols; Cells; Chimera organism; Chimeric Proteins; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Coupling; Cyclophosphamide/Fluorouracil/Prednisone; Data; Development; Drug Kinetics; Environment; Epinephrine; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; G-Protein-Coupled Receptors; GTP-Binding Proteins; Grant; Green Fluorescent Proteins; Hela Cells; Individual; Institution; Kinetics; Life; Ligands; Manuscripts; Methods; Molecular; Monitor; Nature; Paper; Pattern; Peptides; Play; Proteins; Receptor Activation; Research; Research Personnel; Resources; Role; Side; Signal Transduction; Site; Source; Theophylline; Time; United States National Institutes of Health; Variant; base; extracellular; hydroxyl group; member; millisecond; novel strategies; radioligand; receptor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. G-protein coupled receptors (GPCRs) play a crucial role in the transduction of information from the extracellular environment. We monitored activation of GPCRs by fluorescence resonance energy transfer (FRET) between two variants of the green fluorescent protein (CFP and YFP) inserted into the A2A receptor. However, the tagging with two bulky fluorescent proteins eliminates coupling to adenylyl cyclase and slows down the pharmacokinetics of the receptor. In collaboration with NCMIR, we developed a less disruptive FRET-based approach by replacing one of the fluorescent protein with the much smaller tetracysteine tag, expressed the resulting fusion protein in HeLa and HEK293 cells and studied its behavior during receptor activation. Radioligand binding parameters and adenylylcyclase activation were identical to those of the wild-type A2A-receptor. Real-time monitoring of the activation switch of the A2A-receptor uncovered activation kinetics with at 66-88 msec time constant. This novel approach is a significant improvement to the dual GFP-based approach as it enables us to determine accurate pharmacological parameters of GPCRs in living cells within seconds. While the insertion of two bulky fluorescent proteins (CFP and YFP) disturbed the normal pharmacological kinetics of the receptor and affected the downstream signalling, the replacement of YFP with the smaller tetracysteine peptide allowed us to recover almost completely the activity features of the wild type, untagged version. This approach has a considerable potential for generalization to other members of the GPCRA family and further showcases the advantages of small, genetically encoded molecular tags. A synopsis of these data is presented in the manuscript  A FlAsH-Based Approach to Determine G Protein-Coupled Receptor Activation in Living Cells, (Nature Methods, vol.2, no.3, 171-176, 2005). During the previous funding period we have also completed the characterization of the adenosine antagonist theophylline comparing radioligand binding and FRET data. In collaboration with Jean-Pierre Vilardaga, we have conducted a side study using the alpha 2A-receptor chimera used in the Nature Methods paper. Here we compared different ligands systematically broken down by individual hydroxyl-groups from Nor-epinephrine via Dopamin down to tyramin and catechol, investigated their signal at the receptor site and compared it with the signal they give at the G-protein level. We observed a correlation amongst the receptor activation signal, the substitution pattern of hydroxyl-groups and their activation potential of the G-protein.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 G蛋白偶联受体（GPCR）在细胞外环境的信息转导中起着至关重要的作用。我们通过插入A2A受体的绿色荧光蛋白（CFP和YFP）之间的两个变体之间的荧光共振能量转移（FRET）监测GPCR的激活。但是，使用两种大量荧光蛋白的标签消除了与腺苷酸环化酶的耦合，并减慢了受体的药代动力学。 通过与NCMIR合作，我们通过用较小的四环素标签代替一种荧光蛋白来开发了一种较不破坏性的FRET方法，该方法表达了HELA和HEK293细胞中产生的融合蛋白，并研究了其在受体激活中的行为。放射性聚物结合参数和腺苷酸酶活性与野生型A2A受体相同。 对A2A受体的激活开关的实时监测，以66-88毫秒的时间常数为单位。这种新颖的方法是基于双GFP的方法的重大改进，因为它使我们能够在几秒钟内确定活细胞中GPCR的准确药理参数。虽然插入两种笨重的荧光蛋白（CFP和YFP）却扰乱了受体的正常药理动力学并影响了下游信号，但将YFP替换为较小的四环肽肽替换YFP使我们几乎完全恢复了野生型的活性特征，即野生型，即野生型的活性特征。这种方法具有对GPCRA家族其他成员的泛化潜力，并进一步展示了小型，遗传编码的分子标签的优势。这些数据的提要在手稿中提出了一种基于闪光灯的方法，以确定活细胞中G蛋白偶联受体激活（自然方法，第2卷，第3期，第3期，171- 176年，2005年）。在上一个资金期间，我们还完成了比较放射性结合和FRET数据的腺苷拮抗剂Themophanter的表征。与Jean-Pierre Vilardaga合作，我们使用了自然方法论文中使用的alpha 2a受体嵌合体进行了辅助研究。在这里，我们比较了不同的配体，这些配体被从非肾上腺素通过多巴胺降低到泰拉蛋白和儿茶酚的单个羟基分解，研究了它们在受体部位的信号，并将其与它们在G蛋白水平上发出的信号进行了比较。我们观察到受体激活信号之间的相关性，羟基的取代模式及其G蛋白的活化潜力。