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

“G蛋白信号调节剂”（RGS）蛋白超家族成员已成为关键的 特异性G蛋白偶联受体（GPCR）信号转导通路的调节剂。通过他们的“GTZ- RGS蛋白使异源三聚体G蛋白α亚基失活， 从而降低GPCR信号转导。将现有的GPCR激动剂与特异性RGS结构域组合 抑制剂应该增强细胞对这些药物的反应。RGS蛋白质的多样性与高度 人类大脑中局部和动态调节的分布使它们成为有吸引力的目标， 中枢神经系统疾病如帕金森病的药物治疗。不幸的是， RGS蛋白GAP活性的分子抑制剂（或激活剂）可公开用于研究。因此 鉴定小分子工具以进一步提高RGS蛋白在特异性GPCR中功能知识 信号通路，并促进识别用于开发RGS蛋白的先导化合物- 定向治疗，我们将修改和验证新的，实时的，基于荧光的测定RGS 用于自动化高通量分子筛选的蛋白质功能：荧光共振能量 - 基于FRET的结合测定，其采用青色荧光蛋白标记的G-α亚基， 黄色荧光蛋白标记的RGS蛋白，使用荧光标记的单转换GTP水解测定， 用于无机磷酸盐产生传感器，以及G-α核苷酸结合和水解的测定， 使用氟修饰的核苷酸BODIPY <$FL 2 '-（或-3'）-O-（N-（2-氨乙基）氨基甲酸酯）鸟苷5 '- 三磷酸盐 许多有用的药物通过结合细胞表面的一种特殊类型的蛋白质受体而起作用：G蛋白 偶联受体我们的研究小组发现了一个新的蛋白质家族--RGS蛋白质， 这些受体。我们希望找到一种方法来筛选可以阻止RGS蛋白的新药物化合物 从而使现有的药物发挥更有效的作用。 ¿

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. ¿