ALPHA-RECEPTOR PURIFICATION ROLE IN NEUROTRANSMISSION

α受体纯化在神经传递中的作用

• 批准号: 3399692
• 负责人: ROBERT M GRAHAM
• 金额: $ 22.45万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3399692
• 关键词: affinity chromatography; alpha adrenergic receptor; antireceptor antibody; calcium metabolism; complementary DNA; gene expression; guanosine monophosphate; immunochemistry; laboratory mouse; laboratory rabbit; laboratory rat; molecular cloning; neural transmission; phosphatidylinositols; protein metabolism; protein sequence; protein structure; proteins

The alpha1-adrenergic receptor plays a critical role in sympathetic neuro-transmission mediating responses involved, for example, in circulatory homeostasis, metabolism and central nervous system function, such as vasoconstriction, hepatic glycogenolysis and alterations in locomotor activity. Recent evidence suggests that alpha1-adrenergic receptor signalling occurs via a unique mechanism involving membrane polyphosphoinositide breakdown. Activation of this receptor-linked pathway leads to cellular calcium mobilization and may involve coupling via an as yet uncharacterized guanine nucleotide-binding regulatory protein. We have previously purified the mammalian alpha1-adrenergic receptor to homogeneity and investigated its biophysical and structural properties, both directly and after covalent labeling of the receptor with a specific high-affinity, radioiodinated photoaffinity probe that we developed. The major focus of this application is the isolation of a cDNA clone for the receptor using molecular biology strategies as well as more traditional approaches based on amino acid sequence data determined from internal peptides liberated from the purified receptor protein. The receptor cDNA will then be used to clone the receptor's gene and to perform more detailed investigations of receptor expression and receptor structure-function relationships. These studies will involve the use of molecular biology techniques, reconstitution studies and anti-receptor antibodies. Anti-receptor antibodies will be developed using peptide antigens based on various amino acid sequences determined from the elucidated primary structure of the receptor. Additionally, we propose to test the hypothesis that a guanine nucleotide-binding regulatory protein couples the alpha1-adrenergic receptor to polyphosphoinositide breakdown. Initially, studies will be performed to rigorously define the involvement of such a regulatory protein in alpha:- receptor functioning, and to gain mechanistic insights in receptor- regulatory protein interactions. With the availability of this data, we propose to purify this regulatory protein and to evaluate its molecular characteristics using affinity labeling and reconstitution techniques. When completed, these studies should provide important insights into the biochemical and molecular mechanisms of signal transduction by the alpha1-adrenergic receptor. Using the tools of protein and immunochemistry, as well as molecular biology, new approaches will have been developed for understanding alpha1-adrenergic receptor expression, and for defining the kinetics and stoichiometry of its interaction with cellular effector proteins.

α1-肾上腺素能受体在交感神经系统中起重要作用 神经传递介导反应涉及，例如 循环稳态、新陈代谢和中枢神经系统 功能，如血管收缩、肝糖原分解等 运动活动的改变。 最近的证据表明 α1-肾上腺素能受体信号传导通过独特的机制发生 涉及膜多磷酸肌醇的分解。 激活 这种受体相关途径导致细胞钙动员 并且可能涉及通过尚未表征的鸟嘌呤进行偶联 核苷酸结合调节蛋白。 我们之前已经纯化了 哺乳动物α1-肾上腺素能受体的同质性和 研究了其生物物理和结构特性 直接或在受体共价标记后 特定的高亲和力、放射性碘光亲和探针 发达。 该应用的主要重点是隔离 使用分子生物学策略克隆受体的 cDNA 以及基于氨基酸序列的更传统方法 从纯化后释放的内部肽确定的数据 受体蛋白。 然后受体 cDNA 将用于克隆 受体的基因并进行更详细的研究 受体表达和受体结构功能关系。 这些研究将涉及分子生物学技术的使用， 重建研究和抗受体抗体。 抗受体 抗体将使用基于肽抗原来开发 从阐明的初级氨基酸序列中确定的各种氨基酸序列 受体的结构。 此外，我们建议测试 假设鸟嘌呤核苷酸结合调节蛋白 将 α1-肾上腺素能受体与多磷酸肌醇偶联 分解。 最初，将进行严格的研究 定义这种调节蛋白在α中的参与：- 受体功能，并获得受体的机制见解 调节蛋白相互作用。 随着这个的可用性 数据，我们建议纯化这种调节蛋白并评估 使用亲和标记其分子特征和 重构技术。 完成后，这些研究应 为生物化学和分子生物学提供重要见解 α1-肾上腺素能信号转导机制 受体。 还使用蛋白质和免疫化学工具 随着分子生物学的发展，新的方法将被开发出来 了解 α1 肾上腺素能受体表达，以及 定义其相互作用的动力学和化学计量 细胞效应蛋白。