DEVELOPMENT OF A FLUORESCENCE BASED TESTSYSTEM FOR GPCR ACTIVATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 G蛋白偶联受体(GPCRs)在细胞外环境信息传递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的两个变体(CFP和YFP)之间的荧光共振能量转移(FRET)来监测GPCRs的激活。然而，两个体积庞大的荧光蛋白的标记消除了与腺酰环化酶的偶联，并减缓了受体的药代动力学。在与NCMIR的合作中，我们开发了一种破坏性较小的基于FRET的方法，将其中一个荧光蛋白替换为更小的四半胱氨酸标签，在HeLa和HEK293细胞中表达了由此产生的融合蛋白，并研究了其在受体激活期间的行为。放射性配基结合参数和腺苷环化酶激活与野生型A2A型受体相同。实时监测A2a受体的激活开关发现激活动力学为66-88毫秒的时间常数。这种新的方法是对基于双重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GPCRs的准确药理参数。虽然两个庞大的荧光蛋白(CFP和YFP)的插入扰乱了受体的正常药理动力学并影响了下游信号转导，但用较小的四半胱氨酸肽取代YFP使我们几乎完全恢复了野生型、未标记版本的活性特征。这种方法有相当大的潜力推广到GPCRA家族的其他成员，并进一步展示了小的、基因编码的分子标签的优势。这些数据的概要出现在手稿《确定活细胞中G蛋白偶联受体激活的闪光方法》中(《自然方法》，第2卷，第3期，171-176,2005)。在之前的资助期间，我们还完成了腺苷拮抗剂茶碱的表征，比较了放射性配基结合和FRET数据。与Jean-Pierre Vilardaga合作，我们利用《自然方法》论文中使用的阿尔法2A受体嵌合体进行了一项侧面研究。在这里，我们比较了从去甲肾上腺素到多巴胺再到酪氨酸和儿茶酚的不同配体系统分解的羟基，研究了它们在受体位置的信号，并与它们在G蛋白水平上给出的信号进行了比较。我们观察到G蛋白的受体激活信号、羟基取代方式和它们的激活潜力之间存在相关性。

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.