AFFINITY LABELS FOR INOSITOL POLYPHOSPHATE RECEPTORS

肌醇多磷酸受体的亲和标签

• 批准号: 2267750
• 负责人: GLENN DOWNES PRESTWICH
• 金额: $ 21.87万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2267750
• 关键词: affinity labeling; analog; animal tissue; binding proteins; calcium flux; chemical synthesis; circular dichroism; inositol phosphates; laboratory rabbit; nuclear magnetic resonance spectroscopy; phosphatidylinositols; polyphosphates; protein purification; protein sequence; protein structure function; receptor; receptor binding; second messengers; synthetic peptide

Brain cells respond to neurotransmitters and psychoactive drugs through cell-specific receptor proteins. Studies on the integration of diverse signals in healthy and pathological conditions have focused on nitric oxide, cyclic nucleotide, and phosphoinositide pathways. The second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3, or IP3) binds stereospecifically to domains of membrane receptors to cause aggregation and opening of calcium channels. Several IP3 receptors (IP3R) have been cloned and expressed; establishing functional domains in homologous proteins is ongoing. Receptors and functions have been identified for other inositol polyphosphates and phosphoinostides; to date, however, the structures of proteins and binding domains specifically recognizing IP4, IP5, IP6, and the diphosphates IP7 and lP8 remain undetermined. Photoaffinity labels and immobilized affinity matrices will be developed in this proposal for proteins that bind specific inositol polyphosphates (IPn) from IP3 to IP7. These affinity probes will be prepared either from D-glucose or myo-inositol by stereospecific chemical synthesis of analogs selectively functionalized with a phosphodiester bearing an aminopropyl tether. For binding assays and visualization in electrophoretic gels, high specific activity tritium labels will be incorporated. Furthermore, synthesis of phosphatidyl inositol trisphosphate (PtdIns(3,4,5)P3, or PIP3) analogs, will be pursued to provide radioligands for the study of this new ligand. In addition, new synthetic routes to the inositol polyphosphate diphosphates such as IP7 will be developed. For each ligand prepared, the covalently-modified binding sites will be determined by protein digestion and amino acid sequencing. The structural biology of IPn - binding domain interactions will be explored using tools of molecular biology and biophysical chemistry. The phosphoinositide pathway has a pervasive impact in cellular biology, including calcium signaling, hormone signal transduction, odor and taste perception, neurotransmitter action, energy metabolism, protein phosphorylation, and protein trafficking. The affinity reagents and the binding domain data obtained from these reagents will be important in defining the mechanisms of these ubiquitous and varied molecular switches.

脑细胞通过以下方式对神经递质和精神活性药物做出反应 细胞特异性受体蛋白。多元融合研究 健康和病理条件下的信号集中在硝酸 氧化物、环核苷酸和磷酸肌醇途径。第二个 信使 D-肌醇 1,4,5-三磷酸（Ins(1,4,5)P3 或 IP3）结合 立体特异性地作用于膜受体的结构域以引起聚集 和钙通道的开放。一些 IP3 受体 (IP3R) 已被 克隆和表达；建立同源功能域 蛋白质正在进行中。受体和功能已被确定 其他肌醇多磷酸酯和磷酸肌醇酯；然而，迄今为止， 特异性识别 IP4 的蛋白质和结合域的结构， IP5、IP6以及二磷酸盐IP7和IP8仍未确定。 将开发光亲和标签和固定化亲和基质 在这个关于结合特定肌醇多磷酸的蛋白质的提案中 (IPn)从IP3到IP7。这些亲和探针将由以下物质制备 D-葡萄糖或肌醇通过类似物的立体特异性化学合成 用带有氨基丙基的磷酸二酯选择性官能化 系绳。对于电泳凝胶中的结合测定和可视化，高 将纳入特定活性氚标签。此外， 合成磷脂酰肌醇三磷酸 (PtdIns(3,4,5)P3，或 PIP3) 类似物，将致力于为研究提供放射性配体 这个新的配体。此外，肌醇的新合成路线 将开发IP7等聚磷酸二磷酸盐。对于每个配体 制备后，共价修饰的结合位点将通过以下方式确定 蛋白质消化和氨基酸测序。 IPn的结构生物学 - 将使用分子工具探索结合域相互作用 生物学和生物物理化学。 磷酸肌醇途径对细胞生物学具有普遍影响， 包括钙信号传导、激素信号转导、气味和味道 感知、神经递质作用、能量代谢、蛋白质 磷酸化和蛋白质运输。亲和试剂和 从这些试剂获得的结合域数据将非常重要 定义这些普遍存在且多种多样的分子开关的机制。