FORSKOLIN AND GS ALPHA SENSITIVE SOLUBLE ADENYL CYCLASE

毛喉素和 GS ALPHA 敏感可溶性腺苷酸环化酶

• 批准号: 6094253
• 负责人: WEI-JEN TANG
• 金额: $ 6.32万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6094253
• 关键词: Escherichia coli; G protein; active sites; adenylate cyclase; affinity labeling; cyclic AMP; enzyme induction /repression; enzyme mechanism; enzyme model; forskolin; gene targeting; hybrid enzyme; mutant; nucleic acid sequence; protein purification; site directed mutagenesis; solubility

Cyclic AMP (cAMP) is a key second messenger that controls diverse phenomena, including metabolism, gene transcription, olfaction, heart rate, and memory. Cyclic AMP is synthesized by adenylyl cyclases. The modulation of intracellular cAMP concentration is largely at the level of synthesis. The activities of many adenylyl cyclases are controlled dynamically by a variety of hormones, neurotransmitters, and other regulatory molecules. The fundamental components of this pathway are receptors, heterotrimeric G proteins, and adenylyl cyclases. Since defects in the components involved in regulating cAMP concentration are associated with several human diseases, including thyroid and pituitary tumors, it is important to learn more about the regulation of cAMP. Mammalian adenylyl cyclases have a common structure, including two roughly 40 kDa cytoplasmic domains, punctuated by two intensely hydrophobic stretches. Biochemical and genetic analyses are planned to define the regulatory domain(s) of adenylyl cyclase. I have demonstrated that a construct containing two ligated cytoplasmic domains of adenylyl cyclase (IC/1IIC/2) can be expressed as a soluble enzyme in E. coli and it can rescue the catabolic defect of E. coli strains that lack adenylyl cyclase (delta cya). The soluble adenylyl cyclase, IC/1IIC/2, can be regulated by G/s alpha and forskolin. Soluble adenylyl cyclase will be used as a model system to study the adenylyl cyclase site(s) involved in interactions with G/s alpha or forskolin. Complementation of the catabolic defects of E. coli delta cya will be used to screen the randomly mutagenized IC/1IIC/2 expression library for the mutants that gain adenylyl cyclase activity or regulation by G/s alpha or forskolin. Failure of complementation of E. coli delta cya will be used to screen IC/1IIC/2 mutants that lose the adenylyl cyclase activity or regulation by G/s alpha or forskolin. The sensitivity of these mutant enzymes to G/s alpha and forskolin will be assessed and the interesting mutations will be localized by DNA sequencing. The soluble adenylyl cyclases will be purified to homogeneity. The purified enzyme will be labelled with photoaffinity forskolin analogs to determine the forskolin binding site. Determination of the site of adenylyl cyclase that interacts with G/s alpha will allow for manipulation of the interaction between G/s alpha and certain subtypes of adenylyl cyclase, thus providing a strategy to study the physiological role of the G/s alpha mediated signal that is transmitted through a given type of adenylyl cyclase. Determination of the forskolin binding site of adenylyl cyclase will provide crucial information to aid in the design of more specific and potent forskolin analogs to activate adenylyl cyclases.

环磷酸腺苷（cAMP）是一种关键的第二信使， 现象，包括新陈代谢，基因转录，嗅觉，心脏 速度和内存。 环AMP由腺苷酸环化酶合成。 的 细胞内cAMP浓度的调节主要在 合成. 许多腺苷酸环化酶的活性受到 动态地通过各种激素，神经递质，和其他 调节分子 这条途径的基本组成部分是 受体、异源三聚体G蛋白和腺苷酸环化酶。 以来 参与调节cAMP浓度的组分中的缺陷是 与几种人类疾病有关，包括甲状腺和垂体 在肿瘤中，重要的是要了解更多关于cAMP的调节。 哺乳动物腺苷酸环化酶具有共同的结构，包括两个大致相同的结构。 40 kDa的胞质结构域，被两个强疏水性的 伸展运动 计划进行生化和遗传分析， 腺苷酸环化酶的调节结构域。 我已经证明， 含有腺苷酸环化酶的两个连接的胞质结构域的构建体 (IC/1 IIC/2）在E.大肠杆菌， 挽救E.缺乏腺苷酸环化酶的大肠杆菌菌株 （Δ cya）。 可溶性腺苷酸环化酶IC/1 IIC/2可通过 G/S α和毛喉素。 可溶性腺苷酸环化酶将被用作研究 参与与G/s α相互作用的腺苷酸环化酶位点，或 毛喉素 补充E.大肠杆菌 将用于筛选随机诱变的IC/1 IIC/2表达 获得腺苷酸环化酶活性或调节的突变体的文库 G/S α或毛喉素。 E.大肠杆菌δ cya将用于筛选失去腺苷酸的IC/1 IIC/2突变体 环化酶活性或G/s α或毛喉素的调节。 灵敏度 将评估这些突变酶对G/s α和毛喉素的敏感性， 感兴趣的突变将通过DNA测序来定位。 的 将可溶性腺苷酸环化酶纯化至均一。 纯化的 酶将用光亲和性毛喉素类似物标记以确定 毛喉素结合位点。 腺苷酸环化酶位点的确定 与G/s α相互作用的分子将允许操纵 G/s α与腺苷酸环化酶某些亚型之间的相互作用， 从而为研究G/s α的生理作用提供了一种策略， 通过特定类型的腺苷酸传递的介导信号 环化酶 腺苷酸环化酶毛喉素结合位点的测定 将提供重要的信息，以帮助设计更具体， 有效的毛喉素类似物来激活腺苷酸环化酶。