FORSKOLIN AND GS ALPHA-SENSITIVE SOLUBLE ADENYL CYCLASE

毛喉素和 GS α 敏感的可溶性腺苷酸环化酶

• 批准号: 2771030
• 负责人: WEI-JEN TANG
• 金额: $ 18.67万
• 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/2771030
• 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AMP （cAMP）是控制生物多样性的关键第二信使