DEVELOPMENT OF A FLUORESCENCE BASED TESTSYSTEM FOR GPCR ACTIVATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 G蛋白偶联受体（GPCRs）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A2 A受体的绿色荧光蛋白（CFP和YFP）的两种变体之间的荧光共振能量转移（FRET）监测GPCR的激活。然而，用两个庞大的荧光蛋白标记消除了与腺苷酸环化酶的偶联，并减缓了受体的药代动力学。 与NCMIR合作，我们开发了一种破坏性较小的基于FRET的方法，通过用小得多的四半胱氨酸标签替换其中一种荧光蛋白，在HeLa和HEK 293细胞中表达所得融合蛋白，并研究其在受体激活过程中的行为。放射性配体结合参数和腺苷酸环化酶激活与野生型A2 A受体相同。 A2 A受体激活开关的实时监测揭示了66-88毫秒时间常数的激活动力学。这种新方法是对基于GFP的双重方法的重大改进，因为它使我们能够在几秒钟内确定活细胞中GPCR的准确药理学参数。虽然插入两个庞大的荧光蛋白（CFP和YFP）干扰受体的正常药理学动力学和影响下游信号，YFP与较小的四半胱氨酸肽的替代使我们能够恢复几乎完全的野生型，未标记的版本的活性特征。这种方法具有相当大的潜力，推广到其他成员的GPCRA家族，并进一步展示了小，遗传编码的分子标签的优势。这些数据的概要在手稿中提出  一种基于FlAsH的活细胞G蛋白偶联受体活化检测方法，（Nature Methods，第2卷，第3期，171-176页，2005年）。在前一个资助期间，我们还完成了腺苷拮抗剂茶碱的表征比较放射性配体结合和FRET数据。与Jean-Pierre Vilardaga合作，我们使用Nature Methods论文中使用的alpha 2A受体嵌合体进行了一项副研究。在这里，我们比较了不同的配体系统地打破了个别羟基从去甲肾上腺素通过多巴胺到酪胺和儿茶酚，研究它们在受体位点的信号，并将其与它们在G蛋白水平上给出的信号进行比较。我们观察到的受体激活信号之间的相关性，取代模式的羟基基团和它们的G-蛋白的激活潜力。

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.