EFFECT OF MEMBRANE STRUCTURE ON ADENYLATE CYCLASE

膜结构对腺苷酸环化酶的影响

• 批准号: 3278074
• 负责人: Elliott M Ross
• 金额: $ 25.2万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-08-01 至 1992-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278074
• 关键词: G protein; adenylate cyclase; beta adrenergic agent; beta adrenergic receptor; chromatography; complementary DNA; cow; cysteine; disulfide bond; enzyme mechanism; gene expression; genetic manipulation; guanosine triphosphate; laboratory rabbit; membrane lipids; membrane proteins; membrane structure; point mutation; radiotracer; simian virus 40; site directed mutagenesis; tissue /cell culture; turkeys

I propose continued studies on the molecular structure and the mechanism of regulatory activity of the beta-adrenergic receptor. During the last funding cycle, we studied the beta- adrenergic regulation of the stimulatory GTP-binding protein of the adenylate cyclase system, Gs, using purified Gs and beta- adrenergic receptors that were co-reconstituted into unilamellar phospholipid vesicles. (1) We have cloned the cDNA for the receptor and have began to study the organization of the protein. The cloned DNA will be expressed in yeast to provide larger amounts of receptor for biochemical studies and to allow expression of in vitro mutagenized receptor. Expression in a receptor-deficient mammalian cell line will also be developed to allow in situ physiological study of receptor function. (2) The sites of beta-adrenergic agonist binding and of the recently described regulatory cysteine disulfide will be mapped chemically. Functional effects of altering these structures by site-directed mutagenesis will be analyzed after co- reconstituting recombinant receptor with Gs as previously described. (3) The binding of the receptor to Gs will be studied in the vesicles both by covalent crosslinking and by analyzing an isolated agonist-receptor-Gs complex whose formation precedes rapid guanine nucleotide binding. The binding of receptor to Gs will also be assayed directly in the vesicles by measuring fluorescence energy transfer between covalently attached fluorophores on each protein. (4) We will continue enzymologic studies of the mechanism of receptor-mediated regulation of Gs function. The interdependence of receptor and the beta gamma subunits of Gs will be probed in vesicles containing various alpha:beta gamma stoichiometric ratios. Receptor-stimulated binding and release of GTP, GDP, and GTP gamma S will serve as functional assays for receptor function. (5) We will probe the ability of different phospholipids to maintain the activity of the receptor and to support receptor-Gs coupling. We will also test the hypothesis that the rate of receptor-mediated activation of Gs depends on the diffusion-limited collision of receptor and Gs molecules in the membrane.

我建议对分子结构和 β-肾上腺素能调节活性的机理 受体。 在上一个融资周期中，我们研究了Beta- 刺激GTP结合蛋白的肾上腺素能调节 使用纯化的GS和β- 肾上腺素受体共同构成为Unilamellar 磷脂囊泡。 （1）我们将cDNA克隆为 受体并已经开始研究蛋白质的组织。 克隆的DNA将在酵母中表达以提供更大的 用于生化研究的受体量，并允许 体外诱变受体的表达。 在a中的表达 受体缺陷型哺乳动物细胞系也将开发为 允许对受体功能的原位生理研究。 （2） β-肾上腺素能激动剂结合的部位和最近 描述的调节半胱氨酸二硫化物将被映射 化学。 通过 共同分析位置定向的诱变 像以前一样，用GS重组受体重组受体 描述。 （3）将研究受体与GS的结合 在囊泡中，都通过共价交联和分析 孤立的激动剂受体-GS复合物的形成先前 快速鸟嘌呤核苷酸结合。 受体与GS的结合 还将通过测量直接在囊泡中测定 共价附加的荧光能量转移 每种蛋白质上的荧光团。 （4）我们将继续酶学 研究受体介导的GS调节机制 功能。 受体和β伽玛的相互依存关系 GS的亚基将在包含各种的囊泡中进行探测 alpha：beta伽玛化学计量比。 受体刺激 GTP，GDP和GTP伽玛的绑定和释放将服务 作为受体功能的功能测定。 （5）我们将探测 不同磷脂维持活性的能力 受体并支持受体-GS耦合。 我们还将测试 受体介导的激活速率的假设 GS取决于受体和GS的扩散限制碰撞 膜中的分子。