STRUCTURE OF THE BETA ADRENERGIC RECEPTOR CYCLASE SYSTEM

β 肾上腺素能受体环化酶系统的结构

• 批准号: 6018600
• 负责人: Arnold Eino Ruoho
• 金额: $ 30.58万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018600
• 关键词: Baculoviridae; Escherichia coli; G protein; Sf9 cell line; X ray crystallography; adenylate cyclase; affinity labeling; beta adrenergic receptor; binding proteins; chemical binding; chemical structure function; cyclic GMP; forskolin; gene expression; glycosylation; high performance liquid chromatography; intermolecular interaction; ligands; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; phosphodiesterases; protein purification; protein sequence; radionuclides; receptor coupling; rhodopsin; transducin

The focus of this proposal is to understand the molecular interactions which occur in the various domains of the receptor- G protein-effector coupling system through the use of multiple approaches. Novel antagonist and agonist radioiodinated photoactive compounds which are selective for the catechol portion of the b2AR ligand binding site will be developed. These compounds will be used to photoaffinity label the ligand binding domain of the baculovirus-expressed Sf9 b2-AR. Partial N-terminal or complete sequence will be obtained for [125I] photolabeled peptides using a variety of photoprobes to "map" the binding domain of the b2AR. The "exosite" on the b2AR, which is involved in the mechanism of action of long-acting b2 agonists (i.e., salmeterol), will be identified using novel salmeterol derivatives. Soluble cytoplasmic domains of adenylyl cyclase will be expressed in E. coli and purified. The forskolin, ATP, and inhibitory "p" site on the soluble adenylyl cyclase will be identified using photoactivatable compounds and purification of photolabeled peptides. Photolabeling experiments will also be performed on intact adenylyl cyclase, which will be overexpressed in Sf9 cells. Photoactivatable derivatives of specific domain-interacting peptides derived from the sequences of the b2AR and Gas will be utilized to identify interacting domains between b2AR and Gas, between Gas and adenylyl cyclase, and between rhodopsin and alpha transducin. The crystal structure of the catalytically active soluble IC1 and IIC2 adenylyl cyclase will be determined. The binding site domains for forskolin, nucleotides, and Gas will also be determined in the catalytically active soluble IC1 and IIC2 adenylyl cyclase by generating co-crystals. A component of this work involves expression and purification of large quantities of the IC1 domain and/or construction of catalytically active hybrid IC1. IIC2 molecules which are suitable for crystallography. These experiments will increase our understanding of receptor-G protein-effector coupling systems, such as catecholamine beta-receptors, which function to control autonomic functions, such as heart rate, blood pressure, and neuronal function and metabolic state of liver, adipose, and muscle.

该提案的重点是了解分子 发生在受体各个域的相互作用 G 蛋白-效应器耦合系统通过使用多个 接近。 新型拮抗剂和激动剂放射性碘标记 对儿茶酚有选择性的光活性化合物 b2AR配体结合位点的一部分将被开发。 这些化合物将用于光亲和标记配体 杆状病毒表达的 Sf9 b2-AR 的结合域。 部分的 将获得 [125I] 的 N 末端或完整序列 使用各种光探针“绘制”光标记肽 b2AR的结合域。 b2AR 上的“exosite”， 参与长效b2的作用机制 激动剂（即沙美特罗）将使用新颖的方法进行鉴定 沙美特罗衍生物。 腺苷酸的可溶性胞质结构域 环化酶将在大肠杆菌中表达并纯化。 这 毛喉素、ATP 和可溶性腺苷酸上的抑制性“p”位点 环化酶将使用可光活化的化合物进行鉴定，并且 光标记肽的纯化。 照片标记 实验还将在完整的腺苷酸环化酶上进行， 它将在 Sf9 细胞中过表达。 可光激活 特定结构域相互作用肽的衍生物源自 b2AR 和 Gas 的序列将用于识别 b2AR 和 Gas 之间、Gas 和 Gas 之间的相互作用域 腺苷酸环化酶，以及视紫红质和α转导蛋白之间。 催化活性可溶性IC1的晶体结构 并测定IIC2腺苷酸环化酶。 结合位点 forskolin、核苷酸和 Gas 的结构域也将被 测定催化活性可溶性 IC1 和 IIC2 腺苷酸环化酶通过产生共晶。 这个的一个组成部分 工作涉及大量的表达和纯化 IC1结构域和/或催化活性的结构 混合IC1。 IIC2 分子适合 晶体学。 这些实验将增加我们的 了解受体-G蛋白-效应器偶联系统， 例如儿茶酚胺β受体，其功能是控制 自主神经功能，例如心率、血压等 神经元功能和肝脏、脂肪和代谢状态 肌肉。