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: 2396050
• 负责人: Arnold Eino Ruoho
• 金额: $ 28.84万
• 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/2396050
• 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.

该提议的重点是了解分子 在受体的各个结构域中发生的相互作用 通过使用多个 方法。 新颖的反对者和激动剂放射性固定体 光活性化合物是对儿茶酚的选择性的 将开发B2AR配体结合位点的一部分。 这些化合物将用于照片亲和力标记配体 杆状病毒表达的SF9 B2-AR的结合结构域。 部分的 [125i]将获得N末端或完整序列 使用各种光四剂杆进行“地图”的光标记肽 B2AR的结合域。 B2AR上的“ Exosite”， 这参与了长效B2的作用机理 激动剂（即沙美醇）将使用新颖 Salmeterol衍生物。 腺苷的可溶性细胞质结构域 环化酶将在大肠杆菌中表达并纯化。 这 可溶性adenylyl上的Forskolin，ATP和抑制性“ P”位点 将使用光活化化合物来识别循环酶，并且 光标记肽的纯化。 光标记 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。 光活化 特定域相互作用肽的衍生物 B2AR和气体的序列将用于识别 B2AR与气体之间的相互作用域，气体和气体之间 腺苷酸环化酶，在视紫红质和α透明素之间。 催化活性可溶性IC1的晶体结构 将确定IIC2腺苷酸环化酶。 结合位点 Forskolin，核苷酸和气体的域也将是 在催化活性可溶性IC1和IIC2中确定 通过产生共晶层的腺苷酸环化酶。 一个组成部分 工作涉及大量的表达和纯化 IC1域和/或催化活性的构建 混合IC1。 适用于IIC2分子 晶体学。 这些实验将增加我们的 了解受体-G蛋白质效应偶联系统， 例如儿茶酚胺β受体，该β受体可以控制 自主功能，例如心率，血压和 肝脏，脂肪和代谢状态的神经元功能和代谢状态 肌肉。