ALPHA-RECEPTOR PURIFICATION ROLE IN NEUROTRANSMISSION

α1-肾上腺素能受体在交感神经中起关键作用 神经传递介导的反应，例如，在 循环内稳态、代谢与中枢神经系统 功能，如血管收缩，肝糖原分解和 运动活动的改变。最近的证据表明 α1-肾上腺素能受体信号通过一种独特的机制发生 涉及膜多聚磷脂酰肌醇的分解。激活 这一受体连接的途径导致细胞钙动员 并可能涉及通过迄今尚未表征的鸟嘌呤进行偶联 核苷酸结合调节蛋白。我们之前已经提纯了 哺乳动物α1-肾上腺素能受体的同源性和 研究了它的生物物理和结构特性 直接和在受体的共价标记之后 特定的高亲和力、放射性碘标记的光亲和探针 发展起来的。此应用程序的主要关注点是隔离 利用分子生物学策略获得受体的cDNA克隆 以及更传统的基于氨基酸序列的方法 从纯化后的体内释放的多肽确定的数据 受体蛋白。然后，将使用该受体cDNA来克隆 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， 重组研究和抗受体抗体。抗受体 抗体将使用基于以下条件的多肽抗原来开发 从已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 一种鸟嘌呤核苷酸结合调节蛋白的假设 α1-肾上腺素能受体与聚磷脂酰肌醇的偶联 崩溃了。最初，研究将严格地进行到 定义这种调节蛋白在阿尔法中的参与： 受体的功能，并获得对受体的机械洞察- 调节蛋白质的相互作用。随着这款产品的推出 数据，我们建议纯化这种调节蛋白，并评估 应用亲和标记和分子生物学方法研究其分子特性 重建技术。完成后，这些研究应该会 提供对生物化学和分子生物学的重要见解 α1-肾上腺素能受体的信号转导机制 受体。也使用蛋白质和免疫化学的工具 作为分子生物学，新的方法将被开发出来 了解α1肾上腺素能受体的表达，并为 确定与其相互作用的动力学和化学计量学 细胞效应蛋白。

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.