DEVELOPMENT OF A FLUORESCENCE BASED TESTSYSTEM FOR GPCR ACTIVATION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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受体的两种绿色荧光蛋白（CFP和YFP）变体之间的荧光共振能量转移（FRET）来监测gpcr的激活。然而，两种大体积荧光蛋白的标记消除了与腺苷酸环化酶的偶联，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，通过用更小的四胱氨酸标签替换一个荧光蛋白，在HeLa和HEK293细胞中表达得到的融合蛋白，并研究其在受体激活过程中的行为。放射配体结合参数和腺苷酸环化酶活性与野生型a2a受体相同。对a2a受体激活开关的实时监测揭示了在66-88 msec时间常数范围内的激活动力学。这种新方法是对双重gfp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。虽然两个大体积荧光蛋白（CFP和YFP）的插入扰乱了受体的正常药理学动力学并影响了下游信号传导，但用较小的四半胱氨酸肽替代YFP使我们几乎完全恢复了野生型未标记版本的活性特征。该方法具有推广到GPCRA家族其他成员的巨大潜力，并进一步展示了小的遗传编码分子标签的优势。这些数据的摘要发表在手稿《一种基于flash的方法来确定活细胞中G蛋白偶联受体的激活》（Nature Methods, vol.2, no. 5）中。3, 171-176, 2005)。在之前的资助期内，我们还完成了腺苷拮抗剂茶碱的表征，比较了放射性配体结合和FRET数据。我们与Jean-Pierre Vilardaga合作，使用《自然方法》论文中使用的α 2a受体嵌合体进行了一项附带研究。在这里，我们比较了不同的配体，从去甲肾上腺素到多巴胺，再到酪胺和儿茶酚，它们被单个羟基系统地分解，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g蛋白的受体激活信号、羟基取代模式及其激活电位之间存在相关性。

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.