Real-Time Fluorescence Assay:RGS Domain GAP Activit(RMI)

G蛋白信号调节因子(RGS)-蛋白超家族的成员已成为关键 特定的G蛋白偶联受体(GPCR)信号转导通路的调节物。通过他们的“GTP酶- 加速蛋白质“(GAP)活性，RGS蛋白质失活异三聚体G蛋白α亚基和 从而减少GPCR信号转导。将现有的GPCR激动剂与特定的RGS结构域结合 抑制剂应该会增强细胞对这些药物的反应。高度同源RGS蛋白的多样性 人脑中局部和动态调节的分布使它们成为有吸引力的目标 帕金森氏症等中枢神经系统疾病的药物治疗。不幸的是，不是很小 RGS蛋白GAP活性的分子抑制剂(或激活剂)已公开用于研究。因此，要 寻找小分子工具以进一步了解RGS蛋白在特定GPCR中的功能 信号通路，也有助于鉴定形成RGS蛋白的先导化合物- 指导治疗，我们将修改和验证新的，实时的，基于荧光的RGS分析 高通量分子自动筛选的蛋白质功能：一种荧光共振能量 基于转移(FRET)的结合分析，使用青色荧光蛋白标记的G-α亚基和 黄色荧光蛋白标记RGS蛋白的单周转GTP水解法 用于无机磷生产的传感器，以及G-α核苷酸结合和水解度的测定， 使用氟修饰的核苷酸BODIPY？FL 2‘-(or-3’)-O-(N-(2-aminoethyl)urethane)guanosine 5‘- 三磷酸盐。 许多有用的药物通过结合细胞表面的一种特定类型的蛋白受体发挥作用：G蛋白 偶联受体。我们小组发现了一种新的蛋白质家族--RGS蛋白质，它可以干扰 这些受体。我们希望创造方法来筛选能够阻止RGS蛋白的新药化合物 从而使现有药物发挥更大的作用。 ？

Members of the "regulator of G-protein signaling" (RGS)-protein superfamily have emerged as critical modulators of specific G-protein-coupled receptor (GPCR) signal transduction pathways. Via their "GTPase- accelerating protein" (GAP) activity, RGS proteins deactivate heterotrimeric G-protein alpha subunits and thereby reduce GPCR signal transduction. Combining existing GPCR agonists with specific RGS domain inhibitors should potentiate cellular responses to these drugs. The diversity of RGS proteins with highly localized and dynamically regulated distributions in the human brain makes them attractive targets for pharmacotherapy of central nervous system disorders such as Parkinson's disease. Unfortunately, no small molecule inhibitor (or activator) of RGS protein GAP activity is publicly available for study. Therefore, to identify small molecule tools for further advancing knowledge of RGS protein function in specific GPCR signaling pathways, and also to facilitate identification of lead compounds for developing RGS protein- directed therapeutics, we will modify and validate novel, real-time, fluorescence-based assays of RGS protein function for automated high throughput molecular screening: a fluorescence resonance energy transfer (FRET)-based binding assay that employs cyan fluorescent protein-labeled G-alpha subunits and yellow fluorescent protein-labeled RGS proteins, a single-turnover GTP hydrolysis assay using a fluorescent sensor for inorganic phosphate production, and an assay of G-alpha nucleotide binding and hydrolysis that employs the fluor-modified nucleotide BODIPY¿ FL 2'-(or-3')-O-(N-(2-aminoethyl)urethane)guanosine 5'- triphosphate. Many useful drugs act by binding a particular type of protein receptor on the cell's surface: a G-protein coupled receptor. Ourgroup hasdiscovered a new family of proteins - the RGS proteins - that interfere with these receptors. We wish to create ways to screen for new drug compounds that can stop RGS proteins from interfering and thereby allow existing drugs to act more potently. ¿